Method for detecting, identifying, and quantitating organisms and viruses

ABSTRACT

A method for detecting and quantitating organisms containing R-RNA, t-RNA, other RNA, any member of a large, intermediate or small category of organisms such as any member of a bacterial taxonomic Family, Genus, or Species, and previously unknown organisms. The method comprises contacting the nucleic acid of the organisms whose presence, identification and quantitation are to be determined, with a marked probe comprising nucleic acid molecules complementary to RNA or other nucleic acid sequences, of the said organism, under nucleic acid hybridization conditions, and then determining the degree of hybridization that has occurred. The method may include contacting a sample with an enzyme-detergent mixture to make the nucleic acids of the organism or virus in a sample more readily available for hybridization. The method can also be used to determine the sensitivity of particular groups of organisms to antimicrobial agents, to determine the presence of substances with antimicrobial activity, and to determine the state of growth of microorganisms and other cells.

RELATED APPLICATIONS

This is a division of application Ser. No. 08/199,486, filed Feb. 22,1994 hereby incorporated by reference in its totality (includingdrawings), which is a continuation of Kohne, U.S. Ser. No. 08/179,922,filed Jan. 11, 1994, now abandoned, which is a continuation of07/857,081, filed Mar. 19, 1992, now U.S. Pat. No. 5,288,611, which is acontinuation of 07/584,432, filed Sep. 12, 1990, now abandoned, which isa continuation of 07/464,717, filed Jan. 12, 1990, now abandoned, whichis a continuation of 07/353,208, filed May 17, 1989, now abandoned,which is a continuation of 06/655,365, filed Sep. 4, 1984, nowabandoned, which is a continuation-in-part of 456,729, filed Jan. 10,1983, now abandoned, all entitled "Method for Detecting, Identifying andQuantitating Organisms and Viruses", all hereby incorporated byreference herein.

TECHNICAL FIELD

The invention relates to a method and means for detecting, identifying,and quantitating organisms in biological and other samples. Thus, itrelates to a method for specifically and sensitively detecting andquantitating any organism containing the ribosomal RNA, (hereinafterR-RNA), transfer RNA (hereinafter t-RNA) or other RNA, any members orlarge, intermediate, or small sized categories or taxonomic groups ofsuch organisms; and previously unknown organisms containing R-RNA ort-RNA. The method is capable of detecting the presence of even oneorganism, containing R-RNA or t-RNA.

The invention also involves a method for using specifically producednucleic acids complementary to specific sequences or populations ofdifferent sequences of the RNA class mRNA, or hnRNA, or snRNA, or theclass of RNA sequences (hereinafter precursor specific RNA sequences orbsRNA) which are present only in the precursor mRNA, R-RNA, t-RNA, hnRNAor snRNA molecules, and not in mature mRNA, R-RNA, t-RNA, hnRNA or snRNAmolecules, to detect, identify, and quantitate specific organisms,groups of organisms, groups of eukaryotic cells or viruses in cells.

My invention and the novelty, utility, and unobviousness thereof can bemore clearly understood and appreciated when considered in light of therepresentative background information hereinafter set out, comprisingthis art.

BACKGROUND ART

Each of the cells of all life forms, except viruses, contain ribosomesand therefore ribosomal RNA. A ribosome contains three separatesingle-strand RNA molecules, namely, a large molecule, a medium sizedmolecule, and a small molecule. The two larger R-RNA molecules vary insize in different organisms.

Ribosomal RNA is a direct gene product and is coded for by the R-RNAgene. This DNA sequence is used as a template to synthesize R-RNAmolecules. A separate gene exists for each of the ribosomal RNAsubunits. Multiple R-RNA genes exist in most organisms, many higherorganisms containing both nuclear and mitochondrial R-RNA genes. Plantsand certain other forms contain nuclear, mitochondrial and chloroplastR-RNA genes. For simplicity of discussion hereinafter, the threeseparate R-RNA genes will be referred to as the R-RNA gene.

Numerous ribosomes present in all cells of all life forms. About 85-90percent of the total RNA in a typical cell is R-RNA. A bacteria such aE. coli contains about 10⁴ ribosomes per cell while a mammalian livercell contains about 5×10⁶ ribosomes. Since each ribosome contains one ofeach R-RNA subunit, the bacterial cell and mammalian cell contains 10⁴and 5×10⁶, respectively, of each R-RNA subunit.

Nucleic acid hybridization, a procedure well-known in the art, has beenused in the prior art to specifically detect extremely small or largequantities of a particular nucleic acid sequence, even in the presenceof a very large excess of non-related sequences. Prior art uses ofnucleic acid hybridization are found, for example, in publicationsinvolving molecular genetics of cells and viruses, genetic expression ofcells and viruses; genetic analysis of life forms; evolution andtaxonomy or organisms and nucleic acid sequences; molecular mechanismsof disease processes; diagnostic methods for specific purposes,including the detection of viruses and bacteria in cells and organisms.

Probably the best characterized and most studied gene and gene productare the R-RNA gene and R-RNA, and the prior art includes use ofhybridization of R-RNA and ribosomal genes in genetic analysis andevolution and taxonomic classification of organisms and ribosomal genesequences. Genetic analysis includes, for example, the determination ofthe numbers of ribosomal RNA genes in various organisms; thedetermination of the similarity between the multiple ribosomal RNA geneswhich are present in cells; determination of the rate and extent ofsynthesis of R-RNA in cells and the factors which control them.Evolution and taxonomic studies involve comparing the R-RNA gene basesequence from related and widely different organisms.

It is known that the ribosomal RNA gene base sequence is at leastpartially similar in widely different organisms, and that the DNA of E.coli bacterial ribosomal RNA genes hybridizes well with R-RNA fromplants, mammals, and a wide variety of other bacterial species. Thefraction of the E. coli gene which hybridizes to these other speciesvaries with the degree of relatedness of the organisms. Virtually all ofthe R-RNA gene sequence hybridizes to R-RNA from closely relatedbacterial species, while less hybridizes to R-RNA from distantly relatedbacterial species, and even less with mammalian R-RNA.

As with R-RNAs, t-RNAs are present in all living cells, as well as insome viruses. t-RNA genes are present in chromosomal and plasmid DNAs ofprokaryotes and in the DNA of eukaryotic cells, including the DNA of thenucleus, mitochondria and chloroplasts. Different t-RNA genes for onet-RNA species often exist in a single cell. t-RNA genes of mitochondria,nucleic and chloroplasts are quite different. Many virus genomes includegenes for t-RNAs which are specific to the virus.

t-RNA molecules are direct gene products and are synthesized in thecells using the t-RNA gene as a template. The t-RNA is often synthesizedas part of a larger RNA molecule, and the t-RNA portion is then removedfrom this precursor molecule. After synthesis a fraction of the bases ofthe t-RNA molecule are chemically modified by the cell. A typical t-RNAmolecule contains from 75-85 bases.

Numerous t-RNA molecules are present in all cells of all life forms, andusually about 10 percent of a cell's total RNA is composed of t-RNA, atypical bacterial cell containing about 1.5×10⁵ t-RNA molecules of alltypes. If each different kind of t-RNA is equally represented in abacterial cell then 2500 of each different t-RNA molecule is present ineach cell. A typical mammalian liver cell contains about 10⁸ t-RNAmolecules or an average of about 10⁶ copies per cell of each differentt-RNA type.

During protein synthesis individual amino acids are aligned in theproper order by various specific t-RNAs, each amino acid being orderedby a different t-RNA species. Some amino acids are ordered by more thanone t-RNA type.

There are certain viruses which contain t-RNA genes in their genomes,these genes produce virus specific t-RNA when the virus genome is activein a cell. These t-RNAs can also be present in multiple copies in eachinfected cell.

As with R-RNA genes and R-RNA, the prior art discloses use ofhybridization of t-RNA and t-RNA genes in genetic analysis and evolutionand taxonomic classification of organisms and t-RNA gene sequences.Genetic analysis includes, for example, the determination of the numbersof t-RNA genes in various organisms; the determination of the similaritybetween the multiple t-RNA genes which are present in cells;determination of the rate and extent of synthesis of t-RNA in cells andthe factors which control them. Evolution and taxonomic studies involvecomparing the t-RNA gene base sequence from related and widely differentorganisms.

And as with R-RNA gene base sequences, it is known that an individualt-RNA gene base sequence is at least partially similar in differentorganisms. Total t-RNA shows this same type of relationship and bulkt-RNA from one species will hybridize significantly with t-RNA genes ofa distantly related organism. Rat mitochondrial leucyl-t-RNA hybridizedsignificantly with mitochondria DNA of chicken and yeast (Biochemistry(1975) 14, #10, p. 2037). t-RNA genes have also been shown to be highlyconserved among the members of the bacterial family Enterobacteriaceae.Bulk t-RNA genes from E. coli hybridize well with t-RNA isolated fromspecies representing different genes (J. Bacteriology (1977) 129, #3, p.1435-1439). The fraction of the E. coli t-RNA/gene which hybridizes tothese other species varies with the degree of relatedness of theorganisms. A large fraction of the E. coli t-RNA gene sequencehybridizes to t-RNA from a closely related species while much lesshybridized to R-RNA from distantly related species.

The extent of conservation of the t-RNA gene sequences during evolutionis not as great as that for the R-RNA gene sequences. Nonetheless thet-RNA gene sequences are much more highly conserved than the vast bulkof the DNA sequences present in cells.

The sensitivity and ease of detection of members of specific groups oforganisms by utilizing probes specific for the R-RNA or t-RNA of thatgroup of organisms is greatly enhanced by the large number of both R-RNAand t-RNA molecules which are present in each cell. In addition thehybridization test is made significantly easier since RNA moleculespresent in cells are single-stranded. Thus a denaturation step, such asmust be used for a hybridization test which detects any fraction of cellDNA, is not necessary when the target molecule is RNA. Probes specificfor other classes of cell nucleic acids, besides R-RNA or t-RNA, may beused to specifically detect, identify and quantitate specific groups oforganisms or cells by nucleic acid hybridization. Thus, other classes ofRNA in prokaryotic cells include messenger RNA (hereinafter mRNA), andRNA sequences which are part of a variety of precursor molecules. Forexample R-RNA is synthesized in the bacteria E. coli as a precursormolecule about 6000 bases long. This precursor molecule is thenprocessed to yield the R-RNA subunits (totaling about 4500 bases) whichare incorporated into ribosomes and the extra RNA sequences (1500 basesin total) which are discarded. t-RNA molecules and ribosomal 5S RNA arealso synthesized and processed in such a manner.

In prokaryotic cells infected by viruses there is also virus specificmRNA present. The mRNAs of certain prokaryotic viruses are alsosynthesized as a precursor molecule which contains excess RNA sequenceswhich are trimmed away and discarded.

Many of the prokaryotic mRNAs and virus mRNAs are present up to severalhundred times per cell while thousands of the excess RNA sequencespresent in R-RNA or t-RNA precursor molecules can be present in eachcell.

Eukaryotic cells also contain precursor mRNA, as well as precursor R-RNAand t-RNA, molecules which are larger than the final R-RNA or t-RNAmolecules. In contrast to prokaryotes, many newly synthesized eukaryoticmRNA molecules are much larger than the final mRNA molecule and containexcess RNA sequences which are trimmed away and discarded. Another classof RNA present in eukaryotic cells is heterogeneous nuclear RNA(hereinafter known as hn-RNA), which is a diverse class of RNA whichcontains mRNA precursor molecules (which leave the nucleus for thecytoplasm where protein synthesis occurs) and a large amount of RNAwhich never leaves the nucleus. This fraction also contains a smallfraction of double strand RNA. Eukaryotic nuclei also contain small RNAmolecules called small nuclear RNA (hereinafter snRNA), varying inlength from 100-200 bases.

The abundance, or number of copies per cell, of different mRNA moleculesvaries greatly. This varies from a complex class of mRNA molecules whichare present only 1-2 times per cell, to the moderately abundant class ofRNA molecules which are present several hundred times per cell, to thesuperabundant class of RNA molecules which may be present 10⁴ or moretimes per cell. Many of the RNA sequences present in hnRNA are also veryabundant in each cell. The RNA sequences present in the precursor RNAmolecules for R-RNA, t-RNAs and many mRNAs are also very abundant ineach cell. Individual snRNA sequences are extremely abundant and may bepresent from 10⁴ to 10⁶ times per cell.

Eukaryotic cells are also infected by viruses which produce virusspecific mRNA and in may cases virus specific precursor mRNA moleculeswhich contain RNA sequences not present in the mature mRNA molecule. Theindividual virus specific mRNA and precursor RNA molecules vary inabundance from complex (1-2 copies per cell) to superabundant (around10⁴ copies per cell).

My invention also relates therefore, to a method for specifically andsensitively detecting, identifying and quantitating organisms, as wellas, viruses, present in cells. More particularly, the method is usefulfor sensitively detecting, identifying and quantitating any member ofdifferent sized categories of organisms, eukaryotic cells, viruses, andin some cases previously unknown organisms containing mRNA, hnRNA, snRNAor excess RNA molecules present in R-RNA, t-RNA, mRNA, or hnRNAmolecules.

This invention therefore has broad application to any area in which itis important to determine; the presence or absence of living organisms,or viruses present in cells; the state of genetic expression of, anorganism, cell, virus present in a cell, or groups of cells ofprokaryotic or eukaroytic organisms. Such areas include medical,veterinary, and agricultural diagnostics and industrial andpharmaceutical quality control.

The invention involves a method for using specifically produced nucleicacids complementary to, not only R-RNA and t-RNA, but also to specificsequences or populations of different sequences of the RNA class mRNA orhnRNA or snRNA or the class of RNA sequences (hereinafter known asprecursor specific RNA sequences or psRNA) which are present only in theprecursors mRNA, t-RNA, hnRNA or snRNA molecules and not in mature mRNA,R-RNA, t-RNA, hnRNA or snRNA molecules, to detect, identify andquantitate specific organisms, groups or organisms, groups of eukaryoticcells or viruses in cells, by the process of nucleic acid hybridization.

My invention and the novelty, utility and unknown obviousness thereofcan be more clearly understood and appreciated when considered in thelight of the additional representative background informationhereinafter set out, comprising this art:

1. mRNAs, and psRNAs are present in all organisms and cells, hnRNAs andsnRNAs are present only in eukaryotic cells. Cell organelles whichcontain DNA, including mitochondria and chloroplasts, also contain mRNA,psRNA, R-RNA, and t-RNA.

2. A typical bacterial cell contains more than a thousand genes, thevast majority of which code for a specific protein. A mammalian cellcontains over 10,000 genes each of which can produce RNA. Any gene hasthe potential to produce multiple copies of RNA in a cell. Each specificRNA molecule produced is a direct gene product.

3. Many different mRNA sequences can be present in each organism orcell. The individual cells of a multicelled organism may have differentmRNA sequences present in each cell or in different groups of cells.

Many different hnRNA, psRNA, and snRNA sequences can be present in eachcell or group of cells of a eukaryotic organism.

Cells infected with a specific virus can have present within them avariety of different types of virus specific mRNA and psRNA.

4. The number of copies (hereinafter the abundance) of a specific mRNAin a prokaryotic cell varies from zero to several hundred. The abundanceof a specific psRNA sequence in a prokaryotic organism or cell can be 10to 20 times higher.

The abundance of a specific mRNA molecule in a eukaryotic cell rangesfrom 1-2 to greater than 10⁴ per cell.

The abundance of a specific hnRNA sequence in a eukaryotic cell rangesfrom 1-2 to greater than 10⁴ per cell.

The abundance of a specific snRNA molecule in a eukaryotic cell variesfrom 10⁴ to 10⁶ per cell.

The abundance of a specific psRNA sequence in a eukaryotic cell variesfrom 1-2 to over 10⁴ per cell.

5. In many eukaryotes, RNA of various types is produced from therepeated sequence fractions of the DNA. This can result in a populationof abundant RNA molecules whose sequences are similar but not identicalto one another. A probe complementary to one of these RNA moleculeswill, however, hybridize with all of the other similar RNA molecules.

6. The gene sequences which code for the various individual mRNAs.psRNAs, hnRNAs and snRNAs of viruses and living organisms, have beenconserved to varying degrees through evolution. The vast majority ofthese sequences are much less conserved than t-RNA sequences. Some ofthe sequences, however, are highly conserved. For example the gene whichcodes for histone mRNA is very highly conserved through evolution andthe histone gene sequence is quite similar in widely differentorganisms.

The lack of conservation in the DNA sequences of many of these RNAsallows the production of probes which can readily distinguish betweenclosely related organisms or viruses.

A large number of studies have been done on various mRNAs, hnRNAs,snRNAs and psRNAs (see Gene Expression, Vol. 1 and 2, by B. Lewin, inreferences). These include hybridization of these RNAs in studies ongenetic analysis, regulation and evolution, in prokaryotic andeukaryotic organisms and viruses.

Prior Art Hybridization Procedures

Two basic nucleic acid hybridization procedures are disclosed in theprior art. In one, in solution hybridization, both the probe and samplenucleic acid molecules are free in solution. With the other method thesample is immobilized on a solid support and the probe is free insolution. Both of these methods are widely used and well documented inthe literature. An example of the in solution method is presentedhereinafter in the examples. Also, in the article by Thomas et al.,Proc. Natl. Acad. Sci. USA (1980), 77, p. 520, is an example of theimmobilized method.

The basic components of a nucleic acid hybridization test are:

1. Probe

A marked single strand nucleic acid sequence which is complementary tothe nucleic acid sequences to be detected (that is the targetsequences). As used herein, the target sequence is the total sequence ora sub-sequence of R-RNA, t-RNA, or other RNA.

The probe length can vary from 5 bases to tens of thousands of bases,and will depend upon the specific test to be done. Only part of theprobe molecule need be complementary to the nucleic acid sequence to bedetected (hereinafter the target sequences). In addition, thecomplementarity between the probe and the target sequence need not beperfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base. Aprobe may be composed of either RNA or DNA. The form of the nucleic acidprobe may be a marked single strand molecule of just one polarity ormarked single strand molecule having both polarities present. The formof the probe, like its length, will be determined by the type ofhybridization test to be done.

2. Sample

The sample may or may not contain the target molecule (i.e. the organismof interest). The sample may take a variety of forms, including liquidsuch as water or serum, or solid such as dust, soil or tissue samples.The sample nucleic acid must be made available to contact the probebefore any hybridization of probe and target molecule can occur. Thusthe organism's RNA must be free from the cell and placed under theproper conditions before hybridization can occur. Prior art methods ofin solution hybridization necessitate the purification of the RNA inorder to be able to obtain hybridization of the sample R-RNA with theprobe. This has meant that to utilize the in solution method fordetecting target sequences in a sample, the nucleic acids of the samplemust first be purified to eliminate protein, lipids, and other cellcomponents, and then contacted with the probe under hybridizationconditions. The purifications of the sample nucleic acid takes at leastseveral hours and can take up to a day, depending on the nature andquantity of the sample.

3. Hybridization Method

Probe and sample must be mixed under conditions which will permitnucleic acid hybridization. This involves contacting the probe andsample in the presence of an inorganic or organic salt under the properconcentration and temperature conditions. The probe and sample nucleicacids must be in contact for a long enough time that any possiblehybridization between the probe and sample nucleic acid may occur.

The concentration of probe or target in the mixture will determine thetime necessary for hybridization to occur. The higher the probe ortarget concentration the shorter the hybridization incubation timeneeded.

A nucleic acid hybridization incubation mixture composed of probe andsample nucleic acids must be incubated at a specific temperature for along enough time for hybridization to occur. The length of timenecessary for hybridization to complete depends upon the concentrationof the probe nucleic acid, the concentration of the sample nucleic acidwhich is complementary to the probe, and a basic rate of hybridizationwhich is characteristic of the hybridization conditions used. The basicrate of hybridization is determined by the type of salt present in theincubation mix, its concentration, and the temperature of incubation.Sodium chloride, sodium phosphate and sodium citrate are the salts mostfrequently used for hybridization and the salt concentration used israrely above 1M and sometimes as high as 1.5-2M. The salts mentionedabove yield comparable rates of nucleic acid hybridization when used atthe same concentrations and temperatures, as do the comparablepotassium, lithium, rubidium, and cesium salts. Britten et al. (1974)(Methods in Enzymology, Volume XXIX, part E., ed. Grossman and Moldave;Academic Press, New York, page 364) and Wetmur and Davidson (1968) (J.Molecular Biology, Vol. 31, page 349) present data which illustrates thestandard basic rates of hybridization attained in commonly used salts.The hybridization rates of DNA with RNA vary somewhat from those of DNAhybridizing with DNA. The magnitude of the variation is rarely overtenfold and varies, depending for example, on whether an excess of DNAor RNA is used. See Galau et al. (1977) (Proc. Natl. Acad. Sci. USA, Vol74, #6, pg. 2306).

Certain conditions result in the acceleration of DNA; DNA hybridization.An emulsion of phenol and salt promotes the very rapid hybridization ofDNA when the mixture is agitated. Rate increases several thousand timesfaster than standard DNA hybridization rates are attained with thissystem (Kohne et al., Biochemistry (1977) Vol. 16, p. 532a). DNAhybridization rate acceleration of 50 to 100 fold over the standardrates has also been observed when neutral and anionic dextran polymerswere mixed with single strand DNA in solution (Wetmur, Biopolymers(1975) Vol 14. p. 2517). Neither of these DNA accelerated rateconditions was reporter to accelerate the hybridization rate of DNA: RNAhybridization. I am not aware of any prior art which documents acondition for accelerating the rate of RNA: DNA hybridization.

4. Hybridization Assay

A procedure is need to detect the presence of probe molecules hybridizedto the target molecules. Such a method depends upon the ability toseparate probe which is hybridized to target molecules from probe whichis not hybridized to target molecules. Prior art procedures for assayingin solution hybridization mixtures have been done on sample nucleicacids which are first purified and then contacted with the probe in thehybridization incubation mixture.

Hydroxyapatite (HA) has been used as a standard method for assaying insolution hybridization mixtures for the presence of hybridized probe.Under the proper conditions HA selectively binds hybridized DNA probebut does not bind probe which is not hybridized. Other methods areavailable to assay for hybridized probe. These include S₁ nuclease assaywhich depends on the ability of a specific enzyme to degradenon-hybridized probe to small subunits while the hybridized probe is notdegraded by the enzyme and remains large. The degraded probe can then beseparated from the hybridized probe by a size separation technique.Various methods for assaying for in solution hybridized nucleic acidsare presented in Britten et al. (1974) supra.

The immobilized sample nucleic acid hybridization methods have thehybridization assay built into the hybridization method. These methodsinvolve fixing the sample nucleic acid onto an inert support and thenhybridizing this immobilized nucleic acid with a marked probe which isfree in solution. Hybridization of any probe with the immobilized samplenucleic acid results in the binding of the probe to the sample nucleicacid and therefore the attachment of the probe to the inert support.Non-hybridized probe remains free in solution and can be washed awayfrom the inert support and the hybridized probe. Such a method requiresat least several hours to prepare the sample for nucleic acidhybridization and one to two hours of washing and utilizes large amountsof probe. An advantage of this method is the capability to placemultiple samples on the same inert support and to hybridize and processall the samples at one time. Examples of such an immobilized samplemethod is presented in Analytical Biochemistry (1983) Vol. 128, p. 415,and J. of Infectious Disease (1982) Vol. 145, #6, p. 863.

Making Nucleic Acids Available for Hybridization

In solution nucleic acid hybridization methods have always utilizednucleic acids which have been purified away from other cell components.Nucleic acids in cells and viruses are normally tightly complexed withother cell components, usually protein, and, in this form are notavailable for hybridization. Simply breaking the cell or virus open torelease the contents does not render the nucleic acids available forhybridization. The nucleic acids remain complexed to other cell or viralcomponents even though released from the cell, and may in fact becomeextensively degraded by nucleases which also may be released. Inaddition a marked probe added to such a mix may become complexed to"sticky" cell or viral components and be rendered unavailable forhybridization, or the probe may be degraded by nuclease action.

A variety of prior art methods exist for purifying nucleic acids andseveral of these are described in Maniatis et al., supra. These methodsare all time consuming--one taking an hour is regarded as veryrapid--and require multiple manipulations.

Insofar as I am aware, there is no prior art method for performing insolution nucleic acid hybridization which does not require the use ofsome sort of pre-purification step to make the nucleic acids availablefor hybridization.

The immobilized nucleic acid hybridization methods involve fixing thesample nucleic acid onto an inert support and then hybridizing thisimmobilized nucleic acid with marked probe which is free in solution.The process of fixing the nucleic acids on the intert support provides apurification step effective enough. to make the bound nucleic acidsavailable for hybridization. Most of the non-nucleic acid cell or viralcomponents do not bind to the inert support, and those which do bind doso at a different location than the nucleic acids. Such a methodrequires several hours, at a minimum, to prepare the sample nucleicacids for hybridization. An advantage of this method is the ability toplace multiple samples on the inert support and process them alltogether through the hybridization and the hybridization assay steps.The hybridization assay consists of removing the inert support from thehybridization mixture. Probe which is hybridized to the fixed sampleremains with the inert support while non-hybridized probe remains freein solution.

Thus, while the presence of organisms can be detected by any one of alarge variety of prior art methods, none of these is entirelysatisfactory for one reason or another. Such methods include, e.g.,growth methods, optical detection methods, serologic and immunochemicalmethods, and biochemical methods, as shown below:

Growth Tests:

A large number of different growth tests exist, each useful for thegrowth of a specific organism or group of organisms. Growth tests havethe potential sensitivity to detect one organism. In practice, however,many organisms are difficult or impossible to grow. These tests areusually lengthy, taking from one day, to months, to complete. Inaddition, a very large number of tests would be needed to detect thepresence of any member of a large group of organisms (e.g., allbacteria), assuming that the growth conditions for all members of thegroup are known.

Optical Detection Methods:

Microscopic analysis coupled with differential staining methods is verypowerful, and in many cases, very rapid detection method. A majorproblem with this approach is the detection of specific organisms in thepresence of large quantities of other organisms, for example, theidentification of a specific type of gram negative rod shaped bacteria,in the presence of many different kinds of gram negative rod shapedbacteria. In addition, a large number of tests would be needed to detectthe presence of all members of a large group of organisms (such as thegroup of all bacteria).

Serologic and Immunochemical Methods and Biochemical Tests:

A large number of different types of these tests exist. They are usuallyqualitative, not very sensitive and often require a growth step. A greatmany of these tests would be required to detect all members of a largegroup of organisms.

U.S. Pat. No. 4,358,535 to Falkow et al. discloses a method for thedetection of genetic material, i.e., Genes or Genomes. In this patent aclinical sample or isolate suspected of containing a pathogen istransferred onto an inert porous support, such as a nitrocellulosefilter, and treated in such a way that the cells are localized. Thecells are then treated in such a way as to release their DNA and causeit to couple onto the support. Subsequent treatment causes a separationof the individual DNA strands of the genome. The strands are thencontacted with labeled probes specific for the characteristicpolynucleotide sequence under hybridization conditions. Hybridization ofthe probe to the single stranded polynucleotides from the pathogen isdetected by means of the label.

The method of this patent, for detecting genes or genomes, like theother methods mentioned above does not have the specificity,sensitivity, rapidity or ease of performance of that of my invention. Asummary of comparisons of the Falkow et al. method as disclosed in thepatent and that of applicant's method, as herein disclosed, is set outbelow:

    ______________________________________                                         1. Method of doing hybridization                                             FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         Immobilized method only                                                                          In Solution method                                                            emphasized. Immobilized                                                       method can be used.                                         2. Class of nucleic acid to be detected                                      FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         Genetic material (i.e.,                                                                          Detection of a primary gene                                Genes or Genomes). In                                                                            product (RNA) only. RNA                                    cellular organisms the                                                                           is not present as genetic                                  genetic material is always                                                                       material in cellular                                       DNA.               organisms.                                                  3. Abundance (copies per cell) of nucleic acid sequences to be                   detected                                                                  FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         Virtually all microorganism                                                                      10.sup.4 copies of R-RNA are                               chromosomal genes are pre-                                                                       present per bacterial cell.                                sent only one time per cell.                                                                     About 2 × 13.sup.3 copies of each                    Extrachromosomal genes are                                                                       t-RNA is present in each                                   ususally present 1-3 times                                                                       bacterial cell. Ten to 200 of                              per cell. Ribosomal RNA                                                                          each specific mRNA                                         genes are present 3-6 times                                                                      molecule is present per                                    per cell.          bacterial cell. The numbers                                                   are generally higher in                                                       eukaryotic cells.                                           4. Ability of hybridization method to quantitate nucleic acids               FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         None disclosed     Excellent ability to                                                          quantitate nucleic acids, both                                                DNA and RNA.                                                5. Ability to determine and quantitate the state of genetic                      expression of a cell                                                      FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         Genetic expression cannot be                                                                     Genetic expression can be                                  determined by detecting                                                                          determined and quantitated                                 genetic material.  by using probes which detect                                                  the primary gene products or                                                  RNAs.                                                       6. Relative probability of detecting a false positive during                     diagnosis                                                                 FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         High (detects only specific                                                                      Low (when emphasis is on                                   genes).            detecting RNA).                                             7. Relative sensitivity of detection of nucleic acids                        FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         Good. Nucleic acid hybridi-                                                                      Highly sensitive. From 20                                  zation test are in general                                                                       to 10.sup.4 times more sensitive                           quite sensitive.   than possible with the                                                        approach outlined in                                                          Falkow. RNA is almost                                                         always more abundant than                                                     the genes which make it. The                                                  in solution method also                                                       confers extra sensitivity over                                                the immobilized method.                                     8. Preparation of sample for hybridization test                              FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         Takes from 2-10 hours to                                                                         Takes 1-5 minutes to make                                  immobilize sample nucleic                                                                        sample available for hybridi-                              acids and make them avail-                                                                       zation. RNA in cells is                                    able for hybridization.                                                                          already single stranded. All                               Includes a step for con-                                                                         of the sample nucleic acid is                              verting DNA to single strand                                                                     capable of hybridizing.                                    form. Not all the sample                                                      nucleic acids are capable of                                                  hybridization.                                                                9.  Amount of probe needed                                                    FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         Usually takes 0.01 to 1                                                                          Need 10.sup.-5 to 10.sup.-6                                micrograms of probe in                                                                           micrograms of probe per                                    hybridization mixture.                                                                           sample.                                                    10. Time needed for hybridization to occur                                    FALKOW ET AL. METHOD                                                                             APPLICANT'S METHOD                                         2-20 hours         0.2-0.6 hours                                              ______________________________________                                    

I am not aware of any prior art which teaches my method of detecting thepresence or absence of R-RNA, or of t-RNA characteristics of aparticular group of organisms utilizing nucleic acid hybridizationwherein is used a selected marked nucleic acid molecule complementary toa subsequence of R-RNA from a particular source. Nor am I aware of anyprior art which discloses my method for detecting the presence orabsence of R-RNA in general, or of t-RNA from a particular source, bynucleic acid hybridization using a marked nucleic acid moleculecomplementary to all of the R-RNA, or t-RNA subsequences from a specificsource.

Nor am I aware of any prior art which teaches my method of detecting thepresence or absence of specific sequences or populations of differentspecific sequences of mRNA, psRNA, hnRNA or snRNA to detect, identifyand quantitate specific organisms, groups of organisms, groups ofeukaryotic cells, or specific viruses in cells or a group of specificviruses in cells, by nulceic acid hybridization wherein is used selectedmarked nucleic acid molecules complementary to a subsequence(s), asequence(s) or a population of sequences or subsequences of mRNA, hnRNA,snRNA or psRNA from a particular source.

Nor am I aware of any prior art which teaches my method of detecting thepresence or absence of a nucleic acid characteristic of a particulargroup of organisms or viruses; or of rapidly making available for insolution nucleic acid hybridization with a specific marked probe, thenucleic acids of a particular group of organisms or viruses for anypurpose; or of utilizing an in solution nucleic acid hybridizationmethod which combines a rapid method for making the nucleic acids ofspecific groups of organisms available for hybridization with a specificcomplementary probe, with a method for detecting an organism's nucleicacid by greatly accellerating the rate of in solution hybridization ofthe nucleic acids of an organisms virus and the marked probecomplementary to the organism's or virus's nucleic acid; or ofdetermining the antimicrobial agent sensitivity or antiviral agentsensitivity of a particular group of organisms or viruses; or ofassaying for the presence of antimicrobial or antiviral substances inblood, urine, other body fluids or tissues or other sample; or fordetermining the state of growth of cells; or of detecting microorganismor virus infections; or of rapidly assaying for the presence, in ahybridization mixture, of probe which has hybridized, by contacting themixture with hydroxyapatite under predetermined conditions and thenprocessing the resulting solution in a specific manner.

DISCLOSURE OF THE INVENTION

The present invention provides a method and means for detecting,identifying, and quantitating organisms in biological and other samples,and more particularly to a method for specifically and sensitivelydetecting and quantitating any organism containing the ribosomal RNA,(hereinafter R-RNA), transfer RNA (hereinafter t-RNA) or other RNA; anymembers of large, intermediate, or small sized categories or taxonomicgroups of such organisms; and previously unknown organisms containingR-RNA or t-RNA. The method is capable of detecting the presence of evenone organism, containing R-RNA or t-RNA.

The invention also provides a method for using specifically producednucleic acids complementary to specific sequences or populations ofdifferent sequences of RNA class mRNA, or hnRNA, or snRNA, or the classof RNA sequences (hereinafter precursor specific RNA sequences or psRNA)which are present only in the precursor mRNA, R-RNA, t-RNA, hnRNA orsnRNA molecules, and not in mature mRNA, t-RNA, hnRNA or snRNAmolecules, to detect, identify, and quantitate specific organisms,groups of organisms, groups or eukaryotic cells or viruses in cells.

The invention also provides a method and means having as characterizingqualities: (a) the ability to specifically detect the presence of anyone of a large number of different organisms with a single assayprocedure which also works regardless of the pattern of geneticexpression of any particular organism; (b) the ability to modify thetest to detect only specific categories of organisms, even in thepresence of organisms not in the group of interest; (c) extremely highsensitivity of detection, and ability to detect the presence of oneorganism or cell; (d) the ability to quantitate the number of organismsor cells present; and (e) does not require a growth step.

My invention provides means for detecting the anti-microbial agentsensitivity or antiviral agent sensitivity of a particular group oforganisms or viruses; for assaying the presence of antimicrobial orantiviral substances in blood, urine, other body fluids or tissues orother samples; for determining the state of growth of cells; fordetecting microorganism or virus infections; and for rapidly assayingfor the presence in a hybridization mixture of probe which hashybridized.

As described hereinbefore, R-RNA base sequences are partially similar inwidely different organisms. The more closely related two organisms are,the larger the fraction of the total R-RNA which is similar in the twospecies. The R-RNA sequence of any particular species or organism can beregarded as a series of short R-RNA subsequences, of which onesubsequence is similar in virtually all life forms. Therefore, the R-RNAof almost all life forms must contain this subsequence. A differentsubsequence is similar only in the R-RNA of the members of the Speciesto which that organism belongs. Other subsequences are present in theOrder or organisms that the Species belongs to, and so on.

Because the R-RNA sequences of widely different organisms are at leastpartially similar, the method of my invention, using a probe whichdetects the R-RNA sequences which are similar in widely differentorganisms, can detect the presence or absence of any one or more ofthose organisms in a sample. A marked nucleic acid sequence, orsequences complementary to the R-RNA sequences similar in widelydivergent organisms, can be used as such a probe in nucleic acidhybridization assay.

Because the R-RNA sequences of closely related organisms are moresimilar than those of distantly related organisms, the method of myinvention, which includes using a probe which detects only the R-RNAsequences which are similar in a particular narrow group or organisms,can detect the presence or absence of any one or more of thoseparticular organisms in a sample, even in the presence of manynon-related organisms. These group specific probes can be specific for avariety of different sized categories. One probe might be specific for aparticular taxonomic Genus, while another is specific for a particularFamily or another Genus.

Group specific probes have the ability to hybridize to the R-RNA of onegroup of organisms but not hybridize to the R-RNA of any other group oforganisms. Such a group specific complementary sequence will detect thepresence of R-RNA from any member of that specific group of organismseven in the presence of a large amount of R-RNA from many organisms notbelonging to that specific group.

The total number of R-RNA molecules in a sample is measured by using amarked sequence or sequences complementary to R-RNA and standard excessprobe or excess sample RNA nucleic acid hybridization methodology.

The R-RNA content of cells from a wide variety of organisms is known inthe art. In a broad group of similar organisms, for example bacteria,the amount of R-RNA per cell varies roughly 2-5 fold. Therefore, if thenumber of R-RNA molecules in a sample, and the broad class identity ofthe source of the R-RNA is known, then a good estimate of the number ofcells present in the sample can be calculated. If the broad classidentity is not known it can be determined by hybridizing the sample toa series of selected probes complementary to R-RNA, each of which isspecific for a particular broad category of organisms.

At the present time, the operational detection and quantitation range ofa single assay procedure is from 10⁴ R-RNA molecules (1 bacterium or10⁻² mammailan cells) to about 10¹² R-RNA molecules (10⁸ bacteria or 10⁶mammalian cells) a span of about 10⁸ in cell numbers. A single testcould also be done in such a way as to operationally quantitate from 10³bacteria to 10¹⁰ bacteria. The test is quite flexible in this way.

Because the test for R-RNA is specific and has the ability to detect thepresence of very few organisms there is no need to amplify the numbersof organisms through a growth step.

The practice of that form of my invention which is directed todetermining the presence of an organism which contains R-RNA, in asample which might contain such organism, comprises basically:

a) bringing together the sample, or isolated nucleic acids contained inthat sample, with a probe which comprises marked nucleic acid moleculeswhich are complementary to the R-RNA of all organisms;

b) incubating the resulting mixture under predetermined hybridizationconditions for a predetermined time, and then;

c) assaying the resulting mixture for hybridization of the probe.

When my invention is directed to determining the presence of any memberof a specific category of organisms which contain R-RNA in a samplewhich might contain such organisms, the method comprises:

a) contacting the sample, or the nucleic acids therein, with a probecomprising marked nucleic acid molecules which are complementary only tothe R-RNA of members of the specific category of organisms, but notcomplementary to R-RNA from non-related organisms:

b) incubating the probe and the sample, or the isolated nucleic acidstherein; and

c) assaying the incubated mixture for hybridization of said probe.

My invention can also be used to determine the number of organismspresent in the sample under investigation, by adding to the assaying inthe second above described method, in the event probe hybridization hasoccurred, the step of comparing the quantity of R-RNA present in thesample with the number of R-RNA molecules normally present in individualorganisms belonging to the said specific group.

A.d, of course, included in the variations, within the scope of myinvention, which can be used, is that which comprises, in lieu of thesingle probe of step (a) in the second of the above methods, amultiplicity or battery, of different probes. In such case, eachseparate probe comprises marked nucleic acid molecules which arecomplementary only to the R-RNA of a specific group of organisms andeach probe is specific for a different group of organisms; step (a) isfollowed by incubating each probe-sample mixture under predeterminedhybridization conditions for a pre-determined time, and then assayingeach mixture for hybridization of the probe.

As described hereinbefore t-RNA base sequences are partially similar inwidely different organisms. The more closely related two organisms arethe larger the fraction of t-RNA sequences which are related. Each t-RNAgene sequence can be regarded as a series of short t-RNA subsequences.One subsequence is similar in a large related group of organisms.Another subsequence is similar in an intermediate sized related group oforganisms while a third subsequence is similar in a small related groupof organisms and so on.

Since, also, the t-RNA sequences of widely different organisms are atleast partially similar, the method of my invention, using a probe whichdetects the t-RNA sequences which are similar in widely differentorganisms, can detect the presence or absence of any one or more ofthose organisms in a sample. Thus, a marked nucleic acid sequence, orsequences complementary to the t-RNA sequences similar in widelydivergent organisms, can be used as such a probe in a nucleic acidhybridization assay.

A.d since the t-RNA sequences of closely related organisms are moresimilar than those of distantly related organisms, the method of myinvention, which includes using a probe which detects only the t-RNAsequences which are similar in a particular narrow group of organisms,can detect the presence or absence of any one or more of thoseparticular organisms in a sample, even in the presence of manynon-related organisms. Such group specific probes can be specific for avariety of different sized categories. For example, one probe might bespecific for a particular taxonomic Genus, while another is specific fora particular Family or another Genus.

Group specific probes have the ability to hybridize to the t-RNA of onegroup of organisms but not hybridize to the t-RNA of any other group oforganisms. Such a group specific complementary sequence will detect thepresence of t-RNA from any member of that specific group of organismseven in the presence of a large amount of t-RNA from many organisms notbelonging to that specific group.

In the practice of that form of the invention which is directed todetermining the presence of any member of a specific category oforganisms which contain t-RNA in a sample which might contain suchorganisms, the method comprises:

a) contacting the sample, or the nucleic acids therein, with a probecomprising marked nucleic acid molecules which are complementary only tothe t-RNA of members of the specific category of organisms, but notcomplementary to t-RNA from non-related organisms.

b) incubating the probe and the sample, or the isolated nucleic acidstherein; and

c) assaying the incubated mixture for hybridization of said probe.

My invention can also be used to determine the number of organismspresent in the sample under investigation, by adding to the assaying inthe second above described method in the event probe hybridization hasoccurred, the step of comparing the quantity of t-RNA present in thesample with the number of t-RNA molecules normally present in individualorganisms belonging to the said specific group.

A.d, of course, included in the variations, within the scope of myinvention, which can be used, is that which comprises, in lieu of thesingle probe of step (a) in the second of the above methods, amultiplicity or battery of different probes. In such case, each separateprobe comprises marked nucleic acid molecules which are complementaryonly to the t-RNA of a specific group of organisms and each probe isspecific for a different group of organisms; step (a) is followed byincubating each probe-sample mixture under predetermined hybridizationconditions for a pre-determined time, and then assaying each mixture forhybridization of the probe.

The method and means of my invention are more fully illustrated in thefollowing description of characterizing features of test methods inaccordance with the invention.

Nucleic Acid Hybridization Test Procedures for Detecting andQuantitating RNA

A desirable detection test should: a) be rapid; b) be easy to use; c) behighly sensitive; d) be able to detect and quantitate in just one labassay.

The existence of a nucleic acid probe which will hybridize to R-RNA fromany member of the Genus Legionella, but does not hybridize to R-RNA fromany other source, makes possible a rapid, easy to use, sensitive, insolution detection test which can both detect and quantitate, forexample, Legionella bacteria with the performance of just one laboratoryassay and does not require the purification of nucleic acids from thesample.

A description of the basic aspects of this in solution hybridizationtest procedure follows. While the procedure described is designed fordetecting members of the Genus Legionella, it is obvious that this sametest procedure can be used with the appropriate probe to detect manyother groups or organisms or viruses.

Step 1. Preparing the Sample

Mix the sample with a solution containing a detergent and a proteolyticenzyme. The detergent lyses the bacteria and helps solubilize cellularcomponents while the enzyme destroys the cellular proteins, includingthose enzymes which degrade RNA and DNA. The composition of thedetergent-enzyme mix depends upon the type of detergent and proteolyticenzyme used and the amount and type of sample to be checked. Detergentsused include sodium lauryl sulfate, sarkosyl and Zwittergent, while theenzymes used include Proteinase K and Pronase. A wide variety ofenzymes, solubilizing agents such as chaotropic agents, can be used. Theprobe can also be present in the detergent-enzyme mix added to thesample.

The enzyme-detergent acts very quickly on any Legionella bacteria in thesample. In most cases it is not necessary to incubate the mixture inorder to make the R-RNA available for in solution hybridization with theprobe. In certain cases a short incubation period is needed.

In other situations it is not necessary to include the proteolyticenzyme, and detergent alone will make the R-RNA available for insolution hybridization with the probe.

This approach provides a very rapid and easy method for getting thesample R-RNA into a state where it can hybridize with the probe in an insolution assay. In addition, it allows the hybridization to occur insolution without purifying the sample R-RNA. A key to this method isthat the probe detects Legionella R-RNA. R-RNA is single stranded in thecell and ready to hybridize with the probe once the ribosomal proteinsare removed from the R-RNA. In contrast, to directly detect theribosomal R-RNA DNA (i.e., the gene for R-RNA) or any other DNA sequenceit would be necessary to add a procedure which caused the doublestranded R-RNA gene to separate into two single strands before the probecould hybridize to it.

To the best of my knowledge, there is no prior art concerning the use ofan enzyme-detergent-sample method for making R-RNA, transfer R-RNA, RNAin general or DNA available for in solution hybridization with a probefor the purpose of detecting and quantitating the presence or absence oforganisms in general or a specific group of organisms.

Step 2. Preparing the Hybridization Incubation Mixture

To the sample-enzyme-detergent mix add the probe and sufficient salt toenable hybridization to occur and incubate the resultant mixture at anappropriate temperature. The salt concentration and the temperature ofhybridization incubation combine to determine the criterion. Thecriterion of the incubation condition must be equal to that used toselect the probe or the specificity of the probe may change.

The incubation mixture must be incubated for a long enough time forhybridization to occur. The salt type and concentration determines therate of hybridization which can be attained. Thus, certain salts willpromote very rapid hybridization when used at the proper concentration.An example of such a salt is sodium phosphate. Legionella specific probemixed with purified Legionella R-RNA in 3.0M sodium phosphate buffer(pH=6.8) (hereinafter termed PB) and incubated at 76° C. hybridizes over100 times more rapidly than the same amounts of Legionella probe andR-RNA incubated under standard conditions of 0.72M NaCL, 76° C. (thesetwo conditions are equal in criterion). Other salts can also be used toeffect this hybridization rate acceleration. These include most sodium,ammonium, rubidium, potassium, cesium, and lithium salts.

In 3M PB at 76° C. the hybridization rate of the Legionella specificprobe with Legionella R-RNA present in the PB-enzyme-detergent-sampleprobe mixture is also accelerated by over 100 times over thehybridization rates seen for the standard incubation conditions.Hybridization also occurs between the probe and R-RNA in anenzyme-detergent-sample mixture under standard salt concentrationconditions.

One of the features of the invention, as previously pointed out, is theability to detect very small numbers of organisms by detecting theirR-RNA. This is possible because of the large numbers of R-RNA moleculesin each cell. In Legionella-like organisms 5,000 to 10,000 R-RNAmolecules are present in each individual bacterial cell. One of themajor determinants of the sensitivity of detection which can be achievedwith nucleic acid hybridization is the rate of hybridization which canbe attained. The combination of detection of R-RNA and the use of therate accelerating incubation conditions described above make it possibleto attain extremely high sensitivity of detection of bacteria and otherorganisms in a very short period of time with the use of very smallamounts of sample and probe. An illustrative example of this isdescribed later.

To the best of my knowledge there is no prior art concerning the use ofrate-accelerating systems with in solution hybridization tests fordetermining the presence or absence of an organism or group of organismsby detecting the R-RNA, transfer RNA, other RNA or DNA of the organismsof interest. There is also no prior art of which I am aware concerningthe use of a combination of a rate-accelerating system and theenzyme-detergent-sample-probe mixtures to determine the presence orabsence of a specific organism or virus or group of organisms or virusesby detecting the R-RNA, transfer RNA, or other RNA or DNA of thespecific organism or group of organisms of interest.

Step 3. Assaying the Incubation Mixture for Hybridization of the Probewith Target R-RNA

The signal that the sample contains the target R-RNA molecules (andtherefore the target organism) is the presence of hybridized probe inthe incubation mixture. Thus the incubation mixture must be assayed forthe presence of hybridized probe at the end of the incubation period. Itis desirable that such an assay be easy to perform and rapid. For thisassay the incubation mix is processed by utilizing hydroxyapatite (HA).Under the proper conditions HA binds R-RNA rapidly and completely butdoes not bind the non-hybridized probe molecules. If a probe molecule ishybridized to a target R-RNA molecule the probe also binds to the HAbecause it is physically attached to the R-RNA.

Detection of organisms by detecting their R-RNA is a feature of theinvention. The ability of the HA to bind R-RNA or RNA in general, inseconds, while not binding the probe at all, has allowed the developmentof a hybridization assay method which takes minutes to perform, hasgreat flexibility and which adapts well for handling multiple samples.In addition the sample-detergent-enzyme-probe incubation mixture, can bediluted into the appropriate buffer and directly processed to assay forthe presence of hybridized probe.

HA is known in the art as a substance used for assaying hybridization ofprobes. The assay method described here, which has great advantages overthe prior art uses of HA (Brenner et al., A.alytical Biochm (1969( 28 p.477), can be carried out at room temperature and will work over atemperature range of about 15° C. to about 90° C. It has fewer steps anddoes not require heating at each centrifugation step; it can be carriedout in the presence or absence of detergents such as Zwittergent(Calbiochem, Dan Diego, Calif.) and sodium lauryl sulfate. It is 3-5times faster, and a single assay can be done in 3-5-minutes. It requiresabout 5 times less HA. Detergent concentration can range from 0 to 10%,while the phosphate concentrations can range from 0.1M to 0.2M dependingon the type of assay. The method can also be readily adapted forhandling multiple samples.

Methods other than HA are available to assay for hybridization of theprobe. These include enzyme assays such as the S₁ enzyme method, sizeseparation methods, and a variety of sample immobilization methods. Theprobes discussed here can be used effectively with these and any othermethod of conducing hybridization and hybridization assays.

Procedures for the Production of Group Specific R-RNA Probes

Different approaches can be used to produce group specific probes. Allof these approaches but one, rely differential nucleic acidhybridization methods to identify and purify the group specific probesequences.

Procedure A:

The most useful procedure for producing group specific R-RNA probes usesrecombinant DNA methodology. The steps involved in this procedurefollow: (The specific details of standard DNA recombinant techniques aredescribed in the book, Molecular Cloning, A Laboratory Manual, T.Maniatis et al., Cold Spring Harbor Publication (1982)).

1. Isolate nucleic acid from a specific organism of interest. Standardisolation methods are used.

2. Using this isolated DNA, clone the R-RNA genes of this organism andthen produce large amounts of the ribosomal gene DNA, using standard DNArecombinant technology, as shown in Maniatis et al., supra.

3. Reduce the R-RNA gene DNA to short pieces with restriction enzymesand make a library of these short DNA pieces, using standard DNArecombinant methods, as shown in Maniatis et al., supra.

4. Screen the library and identify a clone which contains a short R-RNAgene sequence which hybridizes only to R-RNA from other members of thetaxonomic Species of the organism of interest. Isolate this clone. Itcontains a Species specific DNA sequence which is complementary only tothe R-RNA of the specific Species to which the organisms of interestbelongs.

Screen the library further and identify and isolate the followingclones: a) a clone which contains a DNA sequence complementary to R-RNAwhich will only hybridize to R-RNA from members of the taxonomic Genusto which the organism of interest belongs; b) a clone which contains aDNA sequence complementary to R-RNA which will only hybridize to R-RNAfrom members of the taxonomic Order to which the organism of interestbelongs; c) a clone which contains a DNA sequence complementary to R-RNAwhich will hybridize only to R-RNA from members of the taxonomic Familyto which the organism of interest belongs; d) a clone which contains aDNA sequence complementary to R-RNA which will hybridize only to R-RNAfrom members of the taxonomic Class to which the organism of interestbelongs; and e) a clone which contains a DNA sequence complementary toR-RNA which will hybridize to R-RNA from as many different life forms aspossible.

The foregoing clone selection scheme is only one of a number of possibleones.

Standard methods of cloning and screening are to be utilized, asdiscussed in Maniatis et al., supra.

5.

a) Produce large amounts of each clone's DNA. From the DNA of eachindividual clone isolate and purify only the DNA sequence which iscomplementary to R-RNA, using one of the many methods existing toaccomplish this, e.g., as in Maniatis et al., supra.

b) In certain instances the total DNA present in a clone is useful as aprobe, in which case the total DNA isolated from the cloning vector isused.

c) In certain other instances, the DNA single strand of the cloningvector which contains the DNA sequence complementary to R-RNA is used asa probe. In such case this strand must be isolated and purified, usingone of the various methods which exist to accomplish this, as describedby Maniatis et al.

6. The probe DNA obtained in 5a, 5b, and 5c must be marked in some wayso that it can be identified in the assay mixture. Many different kindsof markers can be used, the most frequently used marker beingradioactivity. Others include fluorescence, enzymes, and biotin.Standard methods are used for marking the DNA, as set out in Maniatis etal., supra.

7. The group specific R-RNA gene sequence in the cloning vector existsin a double strand state. One of these strands is complementary to R-RNAand will hybridize with it. The other strand will not hybridize to R-RNAbut can be used to produce marked group specific sequences complementaryto R-RNA. This is done by utilizing a DNA or RNA polymerase and nucleicacid precursor molecules which are marked. The enzyme will utilize themarked precursors for synthesizing DNA or RNA using the DNA strand as atemplate. The newly synthesized marked molecule will be complementary toR-RNA and can be used as a group specific probe. The template DNA can beremoved by various established means leaving only single strand markednucleic acid, as described in Maniatis, et al., supra, and the articleby Taylor et al., in Biochemica and Biophys. Acta (1976) 442, p. 324.

Procedure B:

Several enzymes can utilize R-RNA from any source as a template for thesynthesizing of marked DNA complementary to the entire R-RNA sequence.Group specific sequences complementary only to the R-RNA of a particularclass of organisms can be isolated by a hybridization selection process.The fraction of the synthesized marked DNA which hybridizes only to theR-RNA from members of a specific class of organisms can be isolated bystandard hybridization procedures. An example of this process ispresented hereinafter. Such a probe can be produced in sufficientquantities to clone as is described in A. The base sequence of thisclone can be determined by standard methods and the sequence used todirect the production of the probe by chemical synthesis using standardmethods.

Procedure C:

The nucleotide sequences of R-RNA from widely different organisms havebeen determined. Group specific sequences similar to a specific group oforganisms can be identified by comparing these known sequences. Asequence complementary to this group specific R-RNA sequence can then bechemically synthesized and marked, using standard methodology.

Production of Specific Probes Complementary to t-RNA

While different approaches can be used to produce specific t-RNA probes,the same basic approaches described for producing R-RNA probes can beused to produce t-RNA probes. Standard methods are available to isolateindividual t-RNA species and genes and these are well known in the art.The form of the probe may be DNA or RNA, and the length of the probe maybe 12 to thousands of bases long. The probe need not be perfectlycomplementary to the nucleic acid it is specific for, i.e., the targetnucleic acid, and the whole length of the probe need not becomplementary to the target molecule.

Production of Specific Probes Complementary to mRNA, hnRNA, snRNA orpsRNA

The same basic approaches used to produce specific probes complementaryto R-RNA can be used to produce specific probes for specific classes orpopulations of mRNA, hnRNA, snRNA, or psRNA. The methods for isolatingeach class of RNA and further fractionating it are well known in theart. Again the form of the probe may be DNA or RNA, and the length ofthe probe may vary from about 12 to thousands of bases long. Thecomplementary region of the probe need not be perfectly complementary tothe target nucleic acid and the whole length of the probe need not becomplementary to the target molecule.

Isolating Sample Nucleic Acid

Standard prior art methods can be used to isolate nucleic acid from thesamples to be assayed. One standard method of nucleic acid isolation andpurification is presented in the examples section and is also discussedin Maniatas et al., supra.

A new technique for making nucleic acids available in solutionhybridization without performing a purification step is describedhereinafter.

Performing the Nucleic Acid Hybridization

An appropriate amount of marked probe is mixed with the sample nucleicacid. This mixture is then adjusted to a specific salt concentration(NaCl is usually used) and the entire mix incubated at a specifictemperature for a specific time period. At the end of the time periodthe mixture is analyzed by performing a hybridization assay. Manydifferent combinations of salt, solvent, nucelic acid concentrations,volumes, and temperatures exist which allow nucleic acid hybridization.The preferred combination depending on the circumstances of the assay.It is important, however, that the criterion (see "Definitions) of thehybridization steps be identical to criteria used to identify and selectthe group probe. If the criteria of the hybridization step is different,the probe specificity may change. See: "Repeated Sequences in DNA", byBritten and Kohne, Science (1968) 161 p. 529; "Kinetics of Renaturationof DNA", by Wetmur and Davidson, J. Mol. Biol. (1968) 31 p. 349;"Hydroxyapatite Techniques for Nucleic Acid Reassociation", by Kohne andBritten; Procedures in Nucleic Acid Research (1971), eds. Cantoni andDavies, Harper and Row, Vol 2, p. 500.

Two different approaches are used with regard to the amount of probe andsample nucleic acid present in the hybridization mixture. In one, theexcess probe method, there is more probe present than sample nucleicacid, in this case RNA. With the other, the excess RNA method, there ismore R-RNA present than probe. The excess probe method is the method ofchoice for detecting the presence of RNA in unknown samples. It hasseveral advantages which are discussed below. See Tables 1 and 2 forfurther discussion of these two approaches.

Using the excess probe method, the detection and quantitation can bedone with just one lab assay point, if the proper RNA prone isavailable. If the hybridization has gone to completion the amount ofprobe which has hybridized is a direct measure of the amount of RNApresent in the sample. The fact that the probe hybridizes at allindicates that RNA is present, and the amount of probe which hybridizesindicates the amount of RNA present in the sample.

Making sure that the hybridization has gone to completion in a knowntime is important in order to quantitate the RNA. This is readily doneby adding enough probe to ensure that the hybridization goes tocompletion in a selected time period. The more probe added, the fastercompletion is reached. Thus the excess probe method provides a means toensure that the hybridization has gone to completion and to know whenthis has occurred.

In contrast, the detection and quantitation of RNA can't be done withone lab assay point when using the excess R-RNA method. In addition, thetime when the test point should be taken cannot be predicted in theexcess RNA method. Unknown samples with small amounts of RNA willhybridize much more slowly than samples with large amounts of RNA.

The Assay for Hybridization

The signal that RNA of the specific group is in the sample is thepresence of double strand marked probe. Many different methods, welldocumented in the literature, are available for assaying thehybridization mixture for the presence of marked probe in the doublestrand form. The choice of method depends upon the method chosen for thehybridization step, the composition of the hybridization mixture, thetype of marker on the probe and other factors. One commonly used methodis described hereinafter. See also Wetmur and Davidson, Kohne andBritten, and Thomas et al., supra. Also the article by Flavell et al.,Eur. J. Biochem. (1974) 47 p. 535. A.d also, the article by Maxwell etal., Nucleic Acids Research (1978) 5 p. 2033.

In all cases, however, it is important to either assay at or above thesame criterion used for the hybridization reaction or at a criterion atwhich hybridization cannot occur.

Quantitation of Nucleic Acid Sequences by Nucleic Acid Hybridization

The quantity of nucleic acid present in a sample can be determined inseveral ways by nucleic acid hybridization, using methods well known tothe art. The two methods are disclosed hereinafter using the example ofquantitating R-RNA.

It will be understood that the present method is generally applicable inany case where it is necessary to determine the presence or absence oforganisms which contain RNA or DNA and that such includes biologicalsamples such as sputum, serum, tissue swabs, and other animal fluids andtissues as well as industrial and pharmaceutical samples and water.Specific details of the approach will change depending on whether RNA orDNA is being quantitated but the general approach is the same for bothDNA and RNA.

                  TABLE 1                                                         ______________________________________                                        EXCESS SELECTED PROBE METHOD                                                  PROBE: The probe is a specific, selected, marked sequence                     from a member of bacteria group B, which represents 10 percent                of the base sequence of the R-RNA, and hybridizes                             completely with R-RNA from group B bacteria, but does not                     hybridize with R-RNA from other organisms.                                    The probe cannot hybridize with itself.                                       A.  Positive                                                                      Homologous                                                                    Control                                                                       0.1 micro-   Hybridize to                                                                             a)  One percent of                                    gram Probe + completion     the probe will                                    10.sup.-3    and assay      form double                                       micrograms   for double     strand mole-                                      Sample       strand probe   cules.                                            group B                 b)  This is a direct                                  R-RNA                       measure of the                                                                R-RNA sample.                                                                 The number of                                                                 probe molecules                                                               hybridized equals                                                             the number of                                                                 R-RNA molecules                                                               present.                                      B.  Hetero-                                                                       logous                                                                        Control                                                                       0.1 micro-   Hybridize to   The probe does                                    grams Probe +                                                                              completion     not hybridize                                     10.sup.-3 micro-                                                                           and assay      with any R-RNA                                    grams Sample for double     but R-RNA from                                    human R-RNA  strand probe   group B bacteria                              C.  Unknown      Hybridize to                                                                             a)  If no group B                                     Sample       completion     R-RNA is pres-                                    0.1 micro-   and assay      ent, no probe                                     grams Probe +                                                                              for double     will hybridize.                                   Unknown      strand probe                                                                             b)  If group B                                        Sample                      R-RNA is pres-                                                                ent, the probe                                                                will hybridize                                                                and form double                                                               strand mole-                                                                  cules.                                                                    c)  The number of                                                                 probe molecules                                                               hybridized                                                                    equals the                                                                    number of group                                                               B R-RNA mole-                                                                 cules present                                                                 in the sample.                                                            d)  If one percent                                                                of the probe                                                                  hybridizes,                                                                   group B R-RNA                                                                 is present                                                                    since the probe                                                               was selected so                                                               that it would                                                                 hybridize only                                                                with R-RNA from                                                               a group B                                                                     bacteria. Since                                                               the probe will                                                                only hybridize                                                                to group B                                                                    R-RNA, the                                                                    presence of                                                                   other R-RNAs                                                                  will not inter-                                                               fere with the                                                                 detection or the                                                              quantitation of                                                               any bacterial                                                                 R-RNA present.                                                            e)  Using a selected probe                                                        makes it easier to                                                            ensure that the hybrid-                                                       ization is complete. A                                                        selected probe repre-                                                         senting 10 percent of                                                         the R-RNA sequence                                                            will hybridize 10 times                                                       faster than a probe                                                           which is representa-                                                          tive of the total                                                             R-RNA sequence.                                                           f)  The detection of R-                                                           RNA in general is not                                                         possible since the                                                            probe hybridizes only                                                         with group B R-RNA.                                                           The sensitivity of                                                            detection of group B                                                          R-RNA is extremely                                                            high.                                         D.  Summary                                                                       The excess probe method needs                                                 just one assay point in order to                                              detect and quantitate group B                                                 organisms.                                                                ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        EXCESS R-RNA METHOD:                                                          THE USE OF A SELECTED PROBE                                                   PROBE: The probe is specific, selected, marked sequence                       from group B bacteria, which represents one-tenth of the                      R-RNA base sequence of one member of group B. The probe                       hybridizes completely with R-RNA from group B, but does                       not hybridize to R-RNA from other organisms.                                  The probe cannot hybridize with itself.                                       A.  Positive                                                                      Homologous                                                                    Control                                                                       Sample     Hybridize to                                                                             a)  The fraction                                        0.1 micro- completion     of probe which                                      grams Group                                                                              and assay      hybridizes is                                       B R-RNA +  for double     a direct                                            10.sup.-3 micro-                                                                         strand probe   measure of the                                      grams Probe               similarity                                                                    between the                                                                   R-RNA and the                                                                 probe. In this                                                                case 100 per-                                                                 cent of the                                                                   probe can                                                                     hybridize.                                                                b)  This percent-                                                                 age is not a                                                                  measure of the                                                                amount of R-RNA                                                               present. In                                                                   order to deter-                                                               mine this the                                                                 kinetics of                                                                   the reaction                                                                  must be deter-                                                                mined.                                          B.  Hetero-                                                                       logous                                                                        Control                                                                       Sample     Hybridize to   The probe does                                      0.1 micro- completion     not hybridize                                       grams human                                                                              and assay      to non-bacterial                                    R-RNA +    for double     R-RNAs.                                             Probe      strand probe.                                                      10.sup.-3                                                                     micro-                                                                        grams                                                                     C.  Unknown                                                                       Sample                                                                        Sample +   Hybridize to                                                                             a)  If no group B                                       Probe      completion     R-RNA is present                                    10.sup.-3  and assay      in the sample                                       mirco-     for double     there will be                                       grams      strand         no hybridized                                                  probe.         probe.                                                                    b)  If group B                                                                    R-RNA is present                                                              the probe will                                                                be hybridized.                                                            c)  The amount of                                                                 R-RNA can't be                                                                determined from                                                               the percentage                                                                hybridization at                                                              the completion of                                                             the reaction. In                                                              order to determine                                                            this the kinetics                                                             of the hybridiza-                                                             tion must be deter-                                                           mined. Since the                                                              probe will hybri-                                                             dize with only one                                                            type of R-RNA, the                                                            kinetic determina-                                                            tion is simple.                                                           d)  If 100 percent of the                                                         probe has hybridized                                                          with the sample, this                                                         means that group B                                                            R-RNA is present in                                                           the sample. It does                                                           not indicate that only                                                        this R-RNA is present.                                                        Other R-RNAs which do                                                         not hybridize with the                                                        probe may also be pre-                                                        sent in the sample.                                                       e)  If 100 percent of the                                                         probe hybridizes with                                                         the sample, it is                                                             possible to specifi-                                                          cally quantitate the                                                          group B R-RNA in the                                                          present of human R-RNA                                                        by determining the                                                            kinetics of hybridiza-                                                        tion of the probe with                                                        the sample R-RNA. Since                                                       the probe will hybridize                                                      only with group B R-RNA                                                       such a kinetic reaction                                                       will have only one                                                            component, the one from                                                       reacting with group B                                                         R-RNA.                                                                    f)  There are situations                                                          where the hybridiza-                                                          tion can't go to com-                                                         pletion. In this method                                                       the sample R-RNA must                                                         drive the hybridization                                                       to completion, since                                                          only a very small amount                                                      of probe is present.                                                          If there is not suffi-                                                        cient R-RNA in the                                                            sample, the hybridiza-                                                        tion will not be                                                              completed. The inter-                                                         pretation of such a                                                           situation is discussed                                                        below.                                                                        If hybridization of                                                           unknown sample results in                                                     20 percent hybridization of                                                   the probe at the usual                                                        assay time, it is not                                                         possible to tell if the                                                       reaction is complete with                                                     only one time-point.                                                          It is necessary to take                                                       another point at double                                                       the original time to                                                          determine if the hybridiza-                                                   tion value increases. If                                                      it does not increase then                                                     the hybridization is                                                          complete. In this case the                                                    R-RNA is at such low                                                          concentration in the sam-                                                     ple that the probe is in                                                      excess, and the number of                                                     R-RNA molecules present                                                       in the sample is equal to                                                     the number of probe mole-                                                     cules hybridized.                                                             If the hybridization value                                                    is increased, the hybrid-                                                     ization was not over at the                                                   first time-point. A third                                                     time-point must then be                                                       done to determine whether                                                     the reaction was over at                                                      the second time point.                          D.  Summary                                                                       The excess sample R-RNA                                                       method needs multiple assay                                                   points in order to detect and                                                 quantitate, and is much more                                                  time-consuming that the                                                       excess probe method.                                                      ______________________________________                                    

USE OF SELECTED PROBES COMPLEMENTARY TO ONLY A PARTICULAR FRACTION OFTHE R-RNA SEQUENCE FROM A PARTICULAR SOURCE TO DETECT R-RNA VERSUS USEOF UNSELECTED PROBES COMPLEMENTARY TO THE ENTIRE R-RNA SEQUENCE FROM APARTICULAR SOURCE TO DETECT R-RNA

One aspect of my invention which comprises using specifically selectedprobes complementary to only a particular fraction of the R-RNAsequences to detect, quantitate, and identify R-RNA has importantcapabilities and advantages over another aspect of the invention, thatof using unselected probes or sequences complementary to the entireR-RNA sequence to detect R-RNA. The advantages of using a selected probein both excess R-RNA and excess probe hybridization methodologies areset forth below. The problems with using a completely representativeprobe are also presented.

The advantages of using a selected probe over using a completelyrepresentative R-RNA probe, with excess probe hybridization, as well aswith excess R-RNA hybridization, is set out below:

    ______________________________________                                        Advantages of the Excess Probe Hybridization Method                           Problems with Completely                                                                        Advantages of Using                                         Representative R-RNA Probe                                                                      Selected Probes                                             1. R-RNA can be detected in a                                                                   The selected probe can be used                              sample with the excess probe                                                                    to sensitively and specifically                             method but there is no way of                                                                   detect and quantitate the pres-                             determining the type of R-RNA                                                                   ence of a particular R-RNA,                                 present. Thus this probe can't                                                                  in an unknown sample when                                   be used to specifically detect                                                                  used in an excess probe hybrid-                             and quantitate the presence of                                                                  ization method. This can be                                 a particular R-RNA in an un-                                                                    done with just one lab assay,                               known sample, with the excess                                                                   even in the presence of                                     probe hybridized method.                                                                        R-RNA from other organisms.                                 2. As stated above, the excess                                                                  The use of a selected probe                                 probe method cannot be used                                                                     makes it possible to use the                                with this probe to detect or                                                                    excess probe method for                                     quantitate the presence of a                                                                    detecting and quantitating                                  particular R-RNA in a sample.                                                                   the presence of a partic-                                   For this purpose the probe must                                                                 ular R-RNA in an unknown                                    be used in the excess R-RNA                                                                     sample. This greatly                                        method.           simplifies the task.                                        The excess R-RNA method is                                                    much more time consuming, re-                                                 quires much more work, and is                                                 much more complicated than the                                                excess probe method.                                                          Advantages of the Excess R-RNA Hybridization Method                           Problems with Completely                                                                        Advantages of Using                                         Representative Probe                                                                            Selected Probe                                              1. R-RNA can be detected in                                                                     The selected probe can be used                              an unknown sample with this                                                                     to specifically detect and                                  probe, but in many cases there                                                                  quantitate the presence of                                  is no way of determining the                                                                    a particular R-RNA in an                                    type or quantity of R-RNA                                                                       unknown sample in all                                       which is present. Thus in                                                                       situations. This can be                                     many instances the probe                                                                        done even in the presence                                   cannot be used to specific-                                                                     of large amounts of R-RNA                                   ally detect and quantitate                                                                      from other organisms.                                       the presence of a particular                                                  R-RNA in an unknown sample.                                                   2. In many cases the sensi-                                                                     With the selected probe the                                 tivity of detection of a                                                                        presence of R-RNA from other                                specific R-RNA is limited                                                                       organisms does not lower the                                by the presence of R-RNA                                                                        sensitivity of detection of                                 from other organisms.                                                                           a particular R-RNA.                                         3. In many cases where it is                                                                    The detection and quantitation                              possible to detect and quanti-                                                                  of the presence of a particular                             tate the presence of particular                                                                 R-RNA is much easier when a                                 R-RNA, it requires a lot of                                                                     selected probe is utilized.                                 work.                                                                         ______________________________________                                    

Illustrative Embodiments

My invention, illustratively, may be used to determine whether a samplecontains any member of a particular group of living organisms. Themethod, described in the following examples, is a test which may be usedto detect and quantiate the presence of any member or members of aparticular group of bacteria in a sample, even in the presence of largenumbers of organisms which are not members of that particular group.

As set forth in the examples, applicant's method involves firstproducing a group specific R-RNA probe which, at a specific criterion,hybridizes to R-RNA from any member of the specific group of interest,but does not hybridize to R-RNA from any other organisms. The use ofsuch a probe in a nucleic acid hybridization test allows the detectionof any member of that specific group, even in the presence of largenumbers of other organisms.

Examples of the practice of the invention are listed later. Each exampleinvolves the production of a marked nucleic acid probe which willhybridize only with R-RNA from members of a particular group oforganisms.

The basic outline of the method used to produce each probe is asfollows:

1. Produce marked nucleic acid complementary to the R-RNA of a member ofthe group of interest.

2. Hybridize this DNA to R-RNA from a member of the group or groups oforganisms evolutionarily most closely related to the group of organismsfor which the probe is to be specific. Select the fraction of the markednucleic acid which, at a specific criterion, does not hybridize to R-RNAfrom a member of this closest related group of organisms. This fractionis specific for the R-RNA of the organism group of interest and does nothybridize with R-RNA from the most closely related group or groups orany other organism.

EXAMPLE 1 Production of Probe Which Will Hybridize to R-RNA from anyBacteria

In a typical situation, about 10⁶ -10⁷ mammalian cells are grown in atissue culture plate at one time. Bacterial species, especially membersof the taxonomic Class Mollicutes, are known to contaminate tissueculture cells. Members of the Class Mollicutes, unlike most otherbactera, are not readily eliminated by antibiotics, and are troublesomecontaminants of cell cultures. Many different Mollicutes species havebeen detected in tissue culture cells. If just one of these organisms ispresent in the culture plate, it has the potential, even in the presenceof antibiotics, to multiply and produce hundreds of organisms per cell.Such organisms are capable of altering the activity of cells, therebyaffecting the results of various studies and the marketability of cellculture products.

Prior art methods for detecting these organisms involve basicallyqualitative tests, the most commonly used being growth tests,differential staining test and immunologic assays. The growth tests,while quite sensitive, take 3-6 weeks. They have the additionaldisadvantage that many organisms are difficult or impossible to grow.

While the actual detection sensitivity of the staining method is notknown, it is known that more than several organisms per cell have to bepresent.

Immunologic tests are qualitative tests and involve using antibodytoward a particular species. While they can be carried out rapidly, theyare not very sensitive; furthermore, many different antibodies would berequired to detect all types of Mollicutes.

The embodiment of applicant's method described below, is a test whichmay be used to detect and quantitate the presence of any member of thegroup of all bacteria, including the taxonomic Class Mollicutes, todetect the presence of Mollicutes in tissue culture, to detect thepresence of bacteria in tissue which is normally free of bacteria, andto detect the presence of the bacteria even in the presence of largenumbers of mammalian cells.

As set forth in the example, applicant's method involves first making aspecific R-RNA probe which is complementary to R-RNA from any bacteriabut is not complementary to mammalian cell R-RNA. The use of such aprobe in a nucleic acid hybridization test allows the detection of anybacteria type, even in the presence of large numbers of mammalian cells.

A detailed description of this embodiment of the invention follows:

Preparation of R-RNA from Mammalian and Bacterial Cells

Mammalian cells are resuspended in 0.3M Nacl, 0.02M Tris, pH=7.4.Sarkosyl is added to a final concentration of 1 percent to lyse thecells. Immediately upon lysis an equal volume of a 1/1 mixture ofphenol/chloroform is added and the resulting mixture shaken vigorouslyfor 2 minutes. The mixture is then centrifuged (8000×g for 10 minutes)to separate the aqueous and organic phases. The aqueous phase isrecovered, and to this is added another volume of phenol/chloroform.After shaking and centrifugation as above, the aqueous phase is againrecovered. To this is added 2 volumes of 95% ethanol and this mixture isplaced at -20° C. for 2 hours to facilitate precipitation of the nucleicacids. Then the mixture is centrifuged (8000×g, 10 minutes) in order tosediment the precipitate to the bottom of the tube. The liquid is thenremoved. The pelleted nucleic acid is redissolved in water. Thissolution is then made to 0.2 m NaCl, 5×10⁻³ m MgCl₂, 5×10⁻³ M CaCl₂.0.02M Tris (pH=7.4), 50 micrograms per ml of deoxyribonuclease I andincubated at 37° C. for 1 hour. Then add an equal volume ofphenol/chloroform and shake as above. Centrifuge as above and recoverthe aqueous phase. Ethanol precipate the RNA as above. Centrifuge theprecipitate as above and redissolve the pelleted RNA in water. Make thissolution to 2M LiCl and place it at 4° C. for 10-20 hours in order tofacilitate the precipitation of the high molecular weight RNA. Thencentriguge this solution to collect the precipate and redissolve theprecipitate in water. This preparation of RNA contains greater than 95%R-RNA.

Bacterial R-RNA is isolated in a similar manner with the followingexceptions. In those cases where detergent alone does not lyse thebacteria, other means are employed. This usually involves pretreatingthe bacteria with an enzyme (lysozyme) to make them susceptible to lysisby sarkosyl. After lysis of the bacteria the isolation procedure is asdescribed above.

Purified R-RNA is stored at -70° C.

Production of Radioactive DNA Complementary (³ H-cDNA) to MollicutesR-RNA

R-RNA from the species Mycoplasma hominis (M. hominis), a member of thetaxonomic class Mollicutes, is used as a template to synthesizeradioactive cDNA complementary to M. hominis R-RNA.

This cDNA is produced by utilizing the ability of an enzyme, reversetranscriptase, to utilize R-RNA as a template and produce ³ H-cDNAcomplementary (cDNA) to R-RNA. The reverse transcriptase reactionmixture contains the following: 50 mM Tris HCL (pH=8.3), 8 mM MgCl₂, 0.4mM dithiothreitol, 50 mM KCL, 0.1 mM ³ H-deoxythymidinetriphosphate (50curies per millimole), 0.2 mM deoxyadenosinetriphosphate, 0.2 mMdeoxycytidinetriphosphate, 0.2 mM deoxyguanosinetriphosphate, 200micrograms per ml of oligodeoxyribonucleotide primer made from E. coliDNA, 50 micrograms per ml M. hominis R-RNA and 50 units per ml of AMVreverse transciptase. This mixture is incubated at 40° C. for 30minutes. Then ethylene diamine tetraacetic acid (EDTA) (pH=7.3), sodiumdodecyl sulfate (SDS), NaCl and glycogen are added to finalconcentrations of 10⁻² M, 1 percent, 0.3M, and 100 micrograms per mlrespectively. The solution is then mixed with 1 volume ofphenol/chloroform (1/1) and shaken vigorously for 2 minutes, thencentrifuged (8000×g for 10 minutes) and the aqueous phase recovered. Thenucleic acids are precipitated by the addition of 2.5 volumes of 95%ethanol. The precipitate is recovered by centrifugation and redissolvedin H₂ O. This solution contains the template R-RNA and the newlysynthesized ³ H-cDNA.

This solution is then, make to 0.3M NaOH and incubated at 50° C. for 45minutes, and cooled in ice and neutralized with 0.3M HCl. Two andone-half volumes of 95% EtOH are then added to precipitate the remainingnucleic acid and the resulting precipitate redissolved in water. Thissolution is then passed over a Sephadex G-100 column equilibrated to0.3M NaCl, 0.1 percent sarkosyl and the excluded volume recovered. Thissolution is ethanol precipitated and the resulting precipitateredissolved in a small volume of water. The process described in thisparagraph removes the template R-RNA and any remaining precursormaterial from the ³ H-cDNA preparation.

The ³ H-cDNA is then hybridized to M. hominis R-RNA to ensure that it isindeed complementary to this R-RNA. The hybridization mixture consistsof, 0.05 micrograms of single strand ³ H-cDNA, 20 micrograms of M.hominis R-RNA, and 0.48M PB (phosphate buffer) in 1 ml. This mixture isincubated for 0.2 hours at 65° C. and is then diluted to 0.14M PB andpassed over a hydroxyapatite (HA) column equilibrated to 0.14M PB and65° C. ³ H-cDNA hybridized to R-RNA absorbs to the hydroxyapatite (HA)column while non-hybridized ³ H-cDNA passes through the column. Thehybridized ³ H-cDNA is then recovered by elution of the HA column with0.3M PB. This fraction is then dialysed to remove the PB, ethanolprecipitated to concentrate the nucleic acid, centrifuged and thenucleic acid redissolved in water. This solution is then treated withNaOH as described above in order to remove the R-RNA. Afterneutralization, addition of glycogen carrier and concentration byethanol precipitation, the ³ H-cDNA is redissolved in a small volume ofwater. This solution contains only ³ H-cDNA which is complementary to M.hominis R-RNA.

Selection of ³ H-cDNA Which is Complementary to M. hominis RNA but isnot Complementary to Human R-RNA

The purified ³ H-cDNA is further fractionated by hybridizing it with agreat excess of human R-RNA. The hybridization mixture consists of 0.05micrograms of H-cDNA, and 40 micrograms of human R-RNA in one ml of0.48M PB. This incubated at 68° C. for 1 hour and the mixture is thendiluted to 0.14M PB and passed over HA equilibrated to 55° C. and 0.14MPB. The fraction (about 50% of the total) which does not adsorb to theHA (i.e., ³ H-cDNA not hybridized to human R-RNA) is collected. Thisfraction is repassed over a new HA column under the same conditions.Again the non-adsorbed fraction is collected. This fraction is dialysedto remove the PB, ethanol precipitated to concentrate the nucelic acidand redissolved in water. This solution is treated with NaOH, asdescribed earlier, in order to remove the human R-RNA. Afterneutralization, addition of glycogen carrier, and concentration byethanol precipitation, the ³ H-cDNA is redissolved in a small volume ofwater. This ³ H-cDNA preparation is complementary to M. hominis R-RNAbut is not complementary to human R-RNA.

Hybridization of Selected ⁴ H-cDNA with R-RNA from Different Source

The production of the selected ³ H-cDNA probe allows the detection ofbacteria, including members of the Class Mollicutes in mammalian tissueculture cells and mammalian tissues by detecting the presence ofbacterial R-RNA by nucleic acid hybridization. A necessary requirementof such a test is that the selected probe must not hybridize to R-RNAfrom mammalian cells which do not contain bacteria. That thisrequirement is met is shown in Table 3V.

Table 3, parts II and III shown that the probe will detect all membersof the class Mollicutes and should detect all types of bacteria. Forexample, Legionella p. and E. coli and Bacillus subtilis arerepresentatives of very different bacterial types and the probehybridizes with R-RNA from each of these types. Evolutionaryconsiderations indicate that this probe will hybridize to R-RNA fromvirtually any known or unknown bacteria. This is due to the extremeconservation of the R-RNA nucleotide sequence during evolution.

This selected probe is useful for detecting the presence of a specificClass of bacteria, Mollicutes, in tissue culture cells. In most tissueculture cells antibiotics are present in the growth medium and thisprevents the growth of virtually all bacteria but members of the ClassMollicutes. Thus any contamination of a tissue culture preparation isalmost certain to be due to a member of the Class Mollicutes.

An important aspect is the ability to determine the number of organismspresent. In most cases, cell lines and their products are discarded whencells are shown, by prior art methods, to be contaminated. The abilityto quantitate these organisms makes it possible to make judgements as tothe severity of any effects due to contamination. The degree of acontamination may be very light, and only one organism per 1000 cellspresent. This level of contamination would have very little effect onthe cells and in many situations the cell products need not bediscarded. The decision might be made to retain valuable cell linesuntil they become more heavily contaminated. Quantatitive considerationsare important for judging the importance of any kind of a bacterialcontamination.

                  TABLE 3                                                         ______________________________________                                        Hybridization of Selected Mollicutes .sup.3 H-cDNA                            with R-RNA from Widely Different Sources                                                                Percent                                                                       Hybridization                                                                 of H-CDNA                                                       Source of R-RNA                                                                             with R-RNA                                          ______________________________________                                        I.   Control    A.    No R-RNA added,                                                                             <1%                                            Experiments      Self Reaction of                                                              .sup.3 H-cDNA                                                           B.    Mock R-RNA isolation                                                                        <1%                                                       C.    Human cell RNA                                                                              97%                                                             known to be contam-                                                           inated with M. hominis                                                        R-RNA                                                   II.  Hybridization                                                                            A.    Members of the Order                                         of .sup.3 H-cDNA of Mycoplasmatales                                           with R-RNA 1.    Mycoplasma hominis                                                                          97%                                            from different   (infects humans)                                             species of 2.    Mycoplasma salivarius                                                                       93%                                            taxonomic        (infects humans)                                             Class Molli-                                                                             3.    Mycoplasma hyorhinis                                                                        84%                                            cutes            (infects pigs)                                                          4.    Mycoplasma pulmonis                                                                         82%                                                             (infects mice)                                                          B.    Members of the Order                                                          Acholeplasmataceae                                                      1.    Acholeplasma laidlawii                                                                      52%                                                             isolate #1                                                                    (infects cows, birds,                                                         dogs, house cats, mice,                                                       sheep, pigs, and                                                              primates)                                                               2.    Acholeplasma  53%                                                             laidlawii                                                                     (isolate #2)                                                            C.    Members of the                                                                Order                                                                         Spiroplasma-                                                                  taceae                                                                  1.    SMCA (infects 69%                                                             insects and                                                                   mice)                                                                   2.    Honey bee     68%                                                             (isolated from                                                                honey bee)                                                              3.    Cactus (isolated                                                                            71%                                                             from cactus)                                                            4.    Corn Stunt    69%                                                             (isolated from                                                                corn)                                                                   5.    Corn Stunt    65%                                                             (isolated from                                                                insect)                                                 III. Hybridization                                                                            A.    Member of the Family                                         of .sup.3 H-cDNA Enterobacteraceae                                            with R-RNA 1.    Escherischia coli                                                                           52%                                            from other       (infects mammals)                                            types of   B.    Member of the Family                                         bacteria         Legionellaceae                                               (taxonomic 1.    Legionella    >28%                                           Class            pneumophilia                                                 Schizomytes)     (infects man)                                                           C.    Member of the                                                                 Family                                                                        Micrococcaceae                                                          1.    Micrococcus   50-60%                                                          luteus                                                                  2.    Staphylococcus                                                                              50%                                                             aureus                                                                  D.    Member of the Family                                                          Lactobacillaceae                                                        1.    Streptococcus 50%                                                             faecalis                                                                E.    Member of the Family                                                          Bacillaceae                                                             1.    Bacillus      40%                                                             subtilis                                                IV.  Hybridization                   2%                                            of .sup.3 H-cDNA                                                              with R-RNA                                                                    Yeast                                                                    V.   Hybridization    Human (primate)                                                                              1%                                            of .sup.3 H-cDNA Cow (bovine)   1%                                            with R-RNA       Mouse (rodent)                                                                               1%                                            from mam-        Rat (rodent)   1%                                            mals and a       Hamster (rodent)                                                                             1%                                            bird             Rabbit (lagomorph)                                                                           1%                                                             Chicken (avian)                                                                              1%                                       ______________________________________                                    

Excess R-RNA hybridizations are done at 68° C., 0.48M PB. Hybridizationassays are done with hydroxyapatite at 67° C. in 0.14M PB, 0.005% sodiumdodecyl sulfate. The hybridization exposure is sufficient to ensurecomplete reaction of the ³ H-cDNA with nuclear R-RNA or formitochondrial R-RNA. Non-bacterial R-RNA Cot's of at least 2×10³ arereached in the case of the mammals and bird. A non-specific signal of1-2 percent has been substracted from the hybridization values presentedabove.

Quantitation of R-RNA by Nucleic Acid Hybridization

The amount of bacterial R-RNA present in a sample can be determined bymeasuring the kinetics of hybridization of the selected ³ H-c-DNA probewith the RNA isolated from a tissue and comparing these kinetics tothose of a known standard mixture. This can be done even in the presenceof a large excess of mammalian cell R-RNA since the probe does nothybridize with this R-RNA (see Table 3, V).

For measuring the kinetics, the hybridization mixtures contain, 10⁻⁵ to10⁻⁴ micrograms of ³ H-cDNA and 1 to 10³ micrograms of purified sampleRNA in 0.01 to 0.1 ml of 0.48M PB. This mixture is incubated at 68° C.and aliquots are removed, diluted to 0.14M PB and assayed forhybridization at various times after the initiation of the reaction.Hybridization assays are performed using hydroxyapatite as describedearlier. The results obtained are compared to the hybridization of theprobe reacted with standard RNAs containing known amounts of bacterialR-RNA. These standards are mixtures of mammalian cell RNA and knownamounts of a specific bacterial R-RNA.

Detection and Quantitation of Members of the Class Mollicutes in TissueCulture Cells

Table 4 presents data obtained by hybridizing the selected probe withRNA isolated (as described earlier) from three different tissue culturecell sampels. Only cell line number 3 is detectably contaminated and thekinetics of the reaction indicated that about 5×10⁷ bacterial cells arepresent in the tissue culture cells.

                  TABLE 4                                                         ______________________________________                                        Detection and Quantitation of Mollicutes                                      in Tissue Culture Cells                                                                 Hybridi- Percent                                                              zation   Hybridization                                                        Time     of .sup.3 H-cDNA                                                                         Number of Bacteria                              Cell Line (hours)  with RNA   Detected                                        ______________________________________                                        1.  44-2C (rat)                                                                             17       1        None detected                                               40       1        None detected                                 2.  P388 D1M  1.1      1        None detected                                     (mouse)   22.5     1        None detected                                 3.  P388 D1C  0.025    20       5 × 10.sup.7                                (mouse)   16.2     78       (about 1 Mollicute                                                            per mammalian in                                                              cell)                                         ______________________________________                                    

Excess R-RNA Hybridizations are done at 68° C. in 0.48M PB in a volumeof 0.01 to 0.04 ml. Each mixture contains 2×10⁵ micrograms of ³ H-cDNAprobe and 50-200 micrograms of sample RNA.

The following example is another embodiment of the method of myinvention, used for detecting very small numbers, even one trypanosome,in the presence of a large number of blood cells.

The detection of trypanosomes is important since certain members of theprotozoan group Trypanosoma are pathogenic for humans, causing diseasesthat include East African sleeping sickness, West African sleepingsickness, and South American trypanosomiasis. These organisms are largeand have varying characteristic shapes, depending on the stage of thelife cycle. Prior art methods rely mainly on serologic, differentialstaining coupled with microscopic examination and animal inoculationprocedures for detecting these organisms in humans. The serodiagnosticmethods vary in sensitivity and specificity and may be difficult tointerpret. The microscopic methods are most used, however small numbersof the trypanosomes are often difficult to detect in the presence oflarge numbers of blood cells. Animal inoculation is a long and costlyprocedure.

The embodiment of the invention set forth in the following example is amethod which makes it relatively easy to detect the presence of onetrypanosome even when co-present with a large number of blood cells.

EXAMPLE 2 Production of Radioactive DNA Complementary to TrypanosomeR-RNA

Radioactive DNA complementary (³ H-cDNA to trypanosoma brucei R-RNA isproduced in the same way as M. hominis ³ H-cDNA, which is describedabove in detail, except that Trypanosoma b. R-RNA is used as a template.

Selection of Trypanosome ³ H-cDNA Which is Complementary to TrypanosomeR-RNA but is not Complementary to Human R-RNA

This is done in the same way as described earlier for M. hominis exceptthat Trypanosoma b. ³ H-cDNA is hybridized to the human R-RNA.

Use of Selected Trypanosome ³ H-cDNA to Detect and QuantitateTrypanosomes in Human Tissue or Fluid

The production of the selected ³ H-cDNA probe allows the detection andquantitation of trypanosomes in human samples by detecting the presenceof trypanosome R-RNA. A necessary requirement of such a test is that theselected probe must not hybridize to R-RNA from human cells which do notcontain trypanosomes. Table 5 shown that this requirement is met.

                  TABLE 5                                                         ______________________________________                                        Hybridization of Selected Trypanosoma brucei                                  .sup.3 H-cDNA with R-RNA from Different Sources                                                 Percent Hybridization                                       R-RNA Source      of .sup.3 H-cDNA with R-RNA                                 ______________________________________                                        No RNA added      1%                                                          Trypanosome brucei R-RNA                                                                        98%                                                         Bacterial (Mycoplasma hominis)                                                                  1%                                                          R-RNA                                                                         Human R-RNA       1%                                                          Human R-RNA known to be                                                                         98%                                                         contaminated with                                                             Trypanosome brucei                                                            ______________________________________                                    

Excess R-RNA hybridizations are done at 65° C. in 0.48M PB. Reactionsare run for 24 hours and the hybridization exposure is sufficient toensure complete reaction of the human nuclear or mitochondrial R-RNAsand the bacterial R-RNA. Hybridization assays are done withhydroxyapatite at 72° C. in 0.14M PB, 0.005% sodium dodecyl sulfate.

One illustrative probe which I have prepared is specific only formembers of the Genus Legionella. The probe hybridizes to greater thanfifty percent with nucleic acids from diverse members of the GenusLegionella, and does not hybridize significantly with nucleic acids frommammals, yeast and a variety of widely diverse bacterial strains (Table8). The probe hybridizes well even with Legionella species such as L.pneumophila and L. micdadei which show little or no bulk DNArelatedness. Other known Legionella species can be detected by thisprobe used in Table 6; as listed in Table 7. All of the known Legionellaspecies (thus far 23 different species) have been examined and all canbe specifically detected with the probe used in Table 6.

The specificity of this probe makes it possible to detect and quantitatethe presence of Legionella species, even in the presence of largenumbers of non-related bacterial or mammaliam cells. Thus, liver cellsfrom a L. pneumophila infected hamster was assayed for the presence andnumber of Legionella organisms by using the specific probe and wellestablished nucleic acid hybridization procedures. The liver hadpreviously been assayed by a microbiological growth test which indicatedthat about 10⁷ Legionella organisms per gram were present in theinfected liver. Nucleic acid hybridization analysis indicated about1-2×10⁸ Legionella organisms per gram of liver. These results suggestthat the plating efficiency in the growth test is about 5-10 percent.

The specific probe allows the highly sensitive and rapid detection ofLegionella organisms even in the presence of large numbers of mammaliancells. In an assay which took less than 1 day the probe easily detectedthe presence of about 400 Legionella organisms which were mixed with 0.4mg of liver (about 2×10⁵ cells).

                  TABLE 6                                                         ______________________________________                                        Hybridization of Legionella Specific Probe With                               Nucleic Acids from Widely Different Sources                                                              Normalized                                                                    Percent                                                       Nucleic Acid    Probe                                                         Source          Hybridized                                         ______________________________________                                        I.   Controls  1)    No nucleic acid 1                                                       2)    Mock nucleic acid Isolation                                                                   1                                                       3)    L. pneumophila infected                                                                       100                                                           hamster tissue  (Actual                                                                       percent =                                                                     81)                                      II.  Legionella-                                                                             1)    L. bozemanii (WIGA)                                                                           >50                                           ceae      2)    L. dumoffii (TEX-KL)                                                                          >50                                                     3)    L. garmanii (LS-13)                                                                           >50                                                     4)    L. jordanis (BL540)                                                                           >50                                                     5)    L. longbeachae  >50                                                     6)    L. micdadai (HEBA)                                                                            >50                                                     7)    L. MSH9         >50                                                     8)    L. oakridgenis (Oakridge 10)                                                                  >50                                                     9)    L. pneumophila (PHA 1)                                                                        100                                                     10)   L. Lansing 2    >50                                                     11)   L. SC-32C-C8    >50                                      III. Other     1)    Aeormonas hydrophila                                                                          1                                             Bacterial 2)    B. subtilis     1                                             Species   3)    Camplyobacter jejuni                                                                          1                                                       4)    Cytophaga johnsonae                                                                           1                                                       5)    E. coli         1                                                       6)    Flavobacterium breve                                                                          1                                                       7)    Flavobacterium gleum                                                                          1                                                       8)    Flavobacterium  1                                                             meningosepticum                                                         9)    Flavobacterium multivarum                                                                     1                                                       10)   Flavobacterium spiritovarum                                                                   1                                                       11)   Flavobacterium thalophohilum                                                                  1                                                       12)   Flexibacter canadensis                                                                        1                                                       13)   Proteus mirabilis                                                                             1                                                       14)   Proteus rettgeri                                                                              1                                                       15)   Proteus vulgaris                                                                              1                                                       16)   Providencia alicalifaciens                                                                    1                                                       17)   Providencia stuartii                                                                          1                                                       18)   Pseudomonas alcaligenes                                                                       1                                                       19)   Vibrio El Tor   1                                                       20)   Mycoplasma hominis                                                      21)   Mycoplasma hyorhinis                                                                          1                                                       22)   Mycoplasma salivarium                                                                         1                                                       23)   Acholeplasma Laidlawii                                                                        1                                                       24)   Spiroplasma SMCA                                                                              1                                                       25)   Spiroplasma corn stunt                                                                        1                                                       26)   Spiroplasma honey bee                                                                         1                                                       27)   Spiroplasma cactus                                                                            1                                        IV.  Yeast           S. cerevisige   1                                        V.   Mammals         Human           1                                                             Hamster         1                                                             Mouse           1                                        ______________________________________                                    

Excess R-RNA hybridization are done at 76° C., 0.48M PB. Hybridizationassays are done with hydroxyapatite at 72° C. in 0.14M PB, 0.005% sodiumdodecyl sulfate. The hybridization exposure is sufficient to ensurecomplete reaction of the ³ H-cDNA with nuclear R-RNA or formitochondrial R-RNA. Non-bacterial R-RNA Cot's of at least 2×10³ arereached in the case of the mammals and birds.

Table 7 Other Legionella Species Which Can Be Detected By SpecificNucleic Acid Probe of Table 7 Species

L. WA-316

L. WO-44-3C (L. feeleii)

L. Phoenix-1

L. WA-270 A

L. PF-209C-C₂

L. SC 65C3 (ORW)

L. Jamestown 26G1-E2

L. MSH-4

L. Lansing 3

L. SC-18-C9

L. SC-63-C7

L. 81-716 (L. wadsworthii)

EXAMPLE 3 Production of a Probe Which Will Hybridize Only to R-RNA fromMembers of the Genus Legionella

Production of Radioactive DNA Complementary to Legionella R-RNA

R-RNA from the species Legionella pneumophila is used as a template tosynthesize marked (radioactive) cDNA (complementary DNA) complementaryto Legionella pneumophila R-RNA. This cDNA is produce by utilizing theability of an enzyme, reverse transciptase, to utilize R-RNA as atemplate and produce ³ H-cDNA complementary to R-RNA. This is done inthe same way as described for producing M. hominis ³ H-cDNA except thatR-RNA from Legionella pneumophila is used as a template.

Selection of Radioactive Probe which Hybridizes only to R-RNA fromMembers of the Genus Legionella

The purified ³ H-cDNA is fractionated by hybridizing it with a greatexcess of R-RNA from E. coli, Acheolaplasma laidlawaii and Providentiastuartii. The hybridization mixture consists of 0.65-1 micrograms of ³H-cDNA and 20 micrograms of each bacterial R-RNA in 1 ml of 0.48M PB.This mixture is incubated at 76° C. for 1 hour and the mixture is thendiluted to 0.14M PB and passed over HA equilbrated to 72° C., 0.14M PB.The fraction of ³ H-cDNA which does not adsorb to the HA (i.e., the ³H-cDNA not hybridized to the R-RNA) is collected. This fraction is thenpassed over HA under the same conditions as above and again thenon-adsorbed fraction is collected. This ³ H-cDNA is then concentratedand again hybridized with bacterial R-RNA as described above. Thenon-adsorbed fraction is collected and concentrated and then hybridizedwith bacterial R-RNA for a third time as described above andfractionated on HA as above. The non-adsorbed fraction is collected,base treated to remove any R-RNA present and concentrated into water.This ³ H-cDNA preparation will hybridize to any member of the Legionellagenus and will not hybridize to R-RNAs from other sources.

Hybridization of Legionella specific ³ H-cDNA Probe with R-RNA and R-RNAGenes from Different Sources

The selected probe allows the detection of any member of the genusLegionella in a sample by detecting the presence of Legionella R-RNA bynucleic acid hybridization. A necessary requirement of such a test isthat the Legionella specific probe must not hybridize to R-RNA fromother sources.

Quantitation of Legionella R-RNA by Nucleic Acid Hybridization ExcessR-RNA method:

The amount of bacterial R-RNA present in a sample can be determined bymeasuring the kinetics of hybridization of the selected ³ H-cDNA probewith the RNA isolated from a tissue sample and comparing these kineticsto those of a known standard mixture. This can be done even in presenceof a large excess of mammalian cell R-RNA since the probe does nothybridize with this R-RNA.

For measuring the kinetics, the hybridization mixture may contain, forexample, 10⁻⁵ to 10⁻⁴ micrograms of ³ H-cDNA and 0.01 to 10³ microgramsof purified sample RNA in 0.01 to 0.1 ml of 0.48M PB. This mixture isincubated at 76° C. and aliquots are removed, diluted to 0.14M PB andassayed for hybridization at various times after the initiation of thereaction. Hybridization assays are performed using hydroxyapatite asdescribed earlier. The results obtained are compared to thehybridization kinetics of the probe reacted with standard RNAscontaining known amounts of bacterial R-RNA. These standards aremixtures of mammalian cell RNA and known amounts of a specific bacterialR-RNA.

Table 8 presents data on the quantitation of Legionella pneumophilapresent in water samples and in an infected hamster liver sample. Thewater samples and the liver samples were titered for the presence of L.pneumophila by standard quantitative growth assays at the Center forDisease Control in Atlanta, Ga.

                  TABLE 8                                                         ______________________________________                                                             Measured by Excess                                                Measured by R-RNA Nucleic                                                     a Growth Assay                                                                            Acid Hybridization                                       ______________________________________                                        L. pneumophila bacteria per gram of infected hamster liver                                ##STR1##                                                                                    ##STR2##                                            L. pneumophila bacteria per ml of water sample                                            ##STR3##                                                                                    ##STR4##                                            ______________________________________                                    

Excess Probe Method

The amount of bacterial R-RNA present in a sample can also be measuredby doing hybridization under conditions where there is an excess of theLegionella specific ³ H-cDNA probe, relative to the amount of LegionellaR-RNA present. This mixture is hybridized to completion. At this pointeach Legionella R-RNA molecule present in the sample is saturated withprobe molecules. By comparing the amount of probe hybridized to theR-RNA to an appropriately constructed standard calibration curve, theamount of R-RNA in a sample can be determined. A good estimate of thetotal number of Legionella pneumophila bacteria present in the samplecan then be calculated by knowing the average number of R-RNA moleculesper L. pneumophila bacterium.

Table 9 presents data on the quantitation of L. pneumophila present inwater samples as determined by the excess probe acceleratedhybridization rate-enzyme-detergent-sample method described in detail ina later section. The water samples were titered for the presence of Lpneumophila by standard quantitative growth assays. These assays takedays to complete while the hybridization assay takes about 1 hour.

                  TABLE 9                                                         ______________________________________                                                Measured by   Measured by the Excess                                          Growth Assay  Probe Method                                            ______________________________________                                        L. pneumophila bacterial per ml water sample                                             ##STR5##                                                                                      ##STR6##                                           ______________________________________                                    

Probe Specific Only for R-RNA from Members of the Genus Legionella

An Analysis of a Water Sample: Accelerated Hybridization Rate Method

1. Preparation of Sample and Hybridization Incubation Mixture: Mix inthe following order as quickly as possible.

a) 9 microliters of sample

b) 2 microliters of enzyme-detergent solution containing 5 milligrams/mlProteinase K, 0.5M Tris (pH=8.1), 8% sodium dodecyl sulfate (SDS), 4%sodium sarcosinate, 0.25M NaCl, 0.016M EDTA, 0.016 EGTA

c) 1 microliter of probe dissolved in water

d) 20 microliters of 4.8M PB

2. Incubate the mixture at 76° for an appropriate time so that thehybridization reaction is complete.

3. The hybridization assay is performed as follows:

a) Add the incubation mixture to one ml of a room temperature solutioncontaining: 0.05 grams hydroxyapatite (HA), 0.054M PB, 0.02% Zwittergent14 (CalBiochem) (hereinafter referred to as Z-14)

b) Shake the mixture for 30 seconds at room temperature, add 5 ml 0.14MPB, 0.02% Z-14, and incubate the mixture at 72° C. for 2 minutes.

c) Centrifuge the solution to pellet the HA. All centrifugations aredone at room temperature. Decant and save the liquid fraction. This iswash #1.

d) Add 6 ml of 0.14M PB, 0.02% Z-14 solution to the pellet. Resuspendthe HA pellet by vortexing it. Centrifuge to pellet the HA and decantand save the liquid fraction. This is wash #2.

e) Repeat step d. This results in wash #3.

f) Add 6 ml 0.03 and resuspend the HA pellet by vortexing. Centrifugethe suspension to pellet the HA and decant the liquid and assay it forthe presence of the probe. This fraction contains the hybridized probe,if any is present.

It is not necessary to elute the hybridized probe from the HA undercertain conditions. Thus, if the probe is marked with a marker which canbe detected in the presence of HA, the pellet from step e can be assayeddirectly for the probe. In the case of a marker such as Iodine-125 thetube containing the HA pellet can be placed directly into a gammadetection machine.

Other modifications can also be made to make the test faster and easier.Thus, the volume and amount of HA used can be scaled down, and thenumber of washes can also be reduced, depending on the situation. Inother instances it may be desirable to increase the volumes of HA ornumber of washes. A variety of salts other than sodium phosphate, andother detergents can also be used in the assay.

B. A.alysis of a Liquid Sample: Standard Hybridization Rate Method

1. Preparation of Sample and Hybridization Inubation mix. Mix in thefollowing order and as quickly as possible.

a) 14 microliters of sample

b) 2 microliters of enzyme-detergent solution described in A.

c) 1 microliter of probe

d) 3 microliters of 3.2M PB, 0.03M EDTA, 0.03M EGTA

2. Incubate the mixture at 76° C. for an appropriate time so thathybridization will complete.

3. The hybridization assay is performed as follows:

a) Add the incubation mix to 1 ml of a solution containing 0.14M PB,0.02% Z-14, 0.05 grams of HA.

b) From this point on the protocol is identical to that described in A.

C. A.alysis of Tissue Sample: Accelerated Hybridization Rate Method

A 10 percent liver homogenate in water is used as the tissue sample.

1. Preparation of Sample and Incubation Mix. Mix as quickly as possiblein the following order.

a) 8 microliters of sample (10% liver homogenate)

b) 3 milliliters of enzyme-detergent mix containing: 8% SDS, 4% sodiumsarcosinate, 0.25M NaCl, 0.016M EDTA, 0.016M EGTA, 0.38M Tris (pH=8.2),195 milligrams/ml of Pronase.

c) 1 microliter of probe specific only for Legionella R-RNA.

d) 20 microliters of 4.8M PB.

2. Incubate the mixture at 76° for an appropriate time to ensure thatthe hybridization reaction is complete.

3. The hybridization assay is performed as described in the section onanalysis of a water sample; Accelerated Rate Method.

D. A.alysis of Tissue Sample: Standard Hybridization Rate Method

A 10 percent liver homogenate in water used as the sample.

1. Preparation of the Sample and Incubation Mix. Mix as quickly aspossible in the following order.

a) 12 microliters of sample.

b) 4 microliters of enzyme-detergent solution described in B.

c) 1 microliter of probe specific only for Legionella R-RNA.

d) 3 microliters of 3.2M PB, 0.03M EDTA, 0.03M EGTA.

2. Incubate the mixture for an appropriate time at 76° C.

3. The hybridization assay is performed as follows.

a) add the incubation mix to 1 ml of a solution containing 0.14M PB,0.02% Z-14, 0.05 grams HA.

b) from this point the assay is identical to that described in A.

A more detailed description of nucleic acid hybridization tests todetect Legionella pneumophila bacteria in water and liver samples ispresented below.

EXAMPLE 4 Rapid Sensitive Detection of Legionella Bacteria in WaterSample: Accelerated Rate Method

1. The following components were mixed as quickly as possible and in thefollowing order.

a) 4.5 microliters of a water sample containing about 10⁵ Legionellapneumophila bacterial per ml. The number of bacteria in the water wasdetermined at the Center of Disease Control in Atlanta by a growth test.

b) 1 microliter of the enzyme-detergent solution described in A.

c) 0.5 microliters of Legionella specific probe. The amount of probeequalled 7.5×10⁻⁶ micrograms.

d) 10 microliters of 4.8M PB. Assembling the hybridization mixture about2 minutes.

2. Incubate the hybridization mixture for 36 minutes at 76° C.

3. Perform the hybridization assay as described in A. This took about 5minutes.

4. Assay the fractions for the presence of the probe. This took about 10minutes.

The number of Legionella bacteria present in the hybridization mixturewas about 500. This number of organisms was detected and quantitated inabout one hour, from start to finish, with the use of less than 10⁻⁵micrograms of probe. Twenty-three percent of the probe hybridized withthe Legionella R-RNA in this test which was designed to be an excessprobe hybridization test. Control tests were done under the sameconditions, one with no bacteria added, and one with about 10⁵ E. colibacteria present in the hybridization mix. In both cases only 1-2percent of the probe behaved as if it were hybridized.

The above test can be modified to assay larger volumes for the presenceof Legionella bacteria. Thus, one ml of a water sample containing 10⁴Legionella bacteria per ml was centrifuged for 30 minutes to pellet thebacteria. A small amount of enzyme-detergent was added to the pellet anda hybridization test was performed on this mixture using the acceleratedrate method and the Legionella bacteria were readily detected. Muchlarger volumes of sample can be centrifuged and other methods ofconcentrating the bacteria, including membrane filtration, can also beused. These modifications make it possible to detect a small number ofbacteria in a large sample volume. Air samples can also be concentratedby methods, including membrane filtration methods, and small numbers ofbacteria can be detected in large volumes of air sample.

EXAMPLE 5 Rapid Sensitive Detection of Legionella Bacteria in a HamsterLiver Sample

1. The following components were mixed as quickly as possible in thefollowing order.

a) 4 microliters of a 10 percent liver homogenate of a hamster liverinfected with Legionella pneumophila. This is equivalent to 400micrograms of liver or about 6×10⁴ liver cells. About 750 Legionellapneumophila were present in this sample.

b) 4 microliters of an enzyme-detergent solution composed of: 45milligrams/ml Proteinase K, 8% SDS, 4% sodium sarcosinate, 0.5M Tris(pH=8.2), 0.008M EDTA, 0.008M EGTA, 0.25M Nacl.

c) 4 microliters of Legionella specific probe. The quantity of probe wasabout 10⁻⁵ micrograms.

2. Incubate the hybridization mixture at 76° C. for 3 hours.

3. Perform the hybridization assay as described in A.

4. Assay the resulting fractions for the presence of probe hybridized toLegionella R-RNA.

The number of Legionella bacteria present in the hybridization mixturewas about 750 and the amount of R-RNA present in this number ofLegionella cells is about 1.1×10⁻⁵ micrograms. The number of liver cellspresent was about 6×10⁴ and the amount of liver R-RNA present was aboutone microgram. Ten percent of the Legionella specific probe hybridized.Control tests were done with unifected liver in the same manner and lessthan one percent of the probe behaved as if hybridized. Examples 4 and 5illustrate only two of the many possible configurations for such a test.Tests utilizing different volumes, salts, detergents, probes, sampletypes, proteolytic enzymes, amounts of HA, incubation periods, organismtypes, amounts of probe, temperatures of incubation, and hybridizationrate accelerating systems can be successfully utilized within thegeneral context of the tests described here. A.y of the R-RNA probes canbe used in a system comparable to those described above. Non R-RNAprobes can also be used to good effect in these systems with someobvious modifications. For example, a test specific for a particular DNAsequence in a specific organism or group of organisms can be doneexactly as described above if a step is included which converts thedouble strand DNA to the single strand form. In other cases differentmodifications of the method must be used. Bacteria such as Mycobacteriaand Bacilli are difficult to break open. A step which breaks open thesebacteria must then be used in conjunction with the method describedabove. A single incubation, in the absence of detergents, with theenzyme lysozyme, will make most Bacillus bacteria susceptible to lysisby detergents, for example. On the other hand, Mycobacteria are verydifficult to lyse and may have to be physical broken open before theycan be tested for.

A modification of the above method can also be used in conjunction withany transfer RNA probe or a probe specific for any other RNA present inan organism.

A step designed to concentrate small numbers of bacteria or other cellsout of large volumes of samples such as air or liquid can also be usedin conjunction with the hybridization test to detect most otherbacterial organisms or other types of organisms.

While I have described above, in detail, the production and use of anucleic acid probe which hybridizes only to nucleic acids from membersof the Genus Legionella, it will be readily apparent to those skilled inthe art from that example and the others, that other probes can beproduced, based on the procedures illustrated above. Thus the methodused to produce such other probes would be as follows:

1. Produce marked nucleic acid complementary to the R-RNA of a member ofthe group of interest.

2. Hybridize this DNA to the R-RNA from a member of the group oforganisms evolutionarily most closely related to the group of organismsfor which the probe is specific. Select the fraction of the markednucleci acid which, at a specific criterion does not hybridize to R-RNAfrom a member of this closest related group of organisms. This fractionis specific for the organism group of interest.

Examples of these are:

a, The production of a marked probe which hybridizes only with R-RNAfrom a member of the bacterial Genus Leptospira and does not hybridizewith R-RNA other sources.

b. The production of a marked probe which hybridizes only with R-RNAfrom a member of the bacterial Genus Mycoplasma and does not hybridizewith R-RNA from other sources.

c. The production of a marked probe which hybridizes only with R-RNAfrom a member of the bacterial Family Enterbacteriaceae and does nothybridize with R-RNA from other sources.

d. The production of a marked probe which hybridizes only with R-RNAfrom a member of the anaerobic group of bacteria and does not hybridizewith R-RNA from other sources.

e. The production of a marked probe which hybridizes only with R-RNAfrom a member of the group Fungi and does not hybridize with R-RNA fromother sources.

f. The production of a marked probe which hybridizes only with R-RNAfrom any member of the Chlamydia group and does not hybridize with R-RNAfrom other sources.

g. The production of a marked probe which hybridizes only with R-RNAfrom any member of the bacterial family Mycobacteriaceae and does nothybridize with R-RNA from other sources.

h. The production of a marked probe which hybridizes R-RNA from anyliving organism.

i. The production of marked probe which hybridizes only with R-RNA fromany mammal and does not hybridize with R-RNA from other sources.

Illustrative Embodiment of the Use of Probes for t-RNA to Detect,Quantitate and Identify Organisms

t-RNA probes may be used in the same manner as the R-RNA probes todetect, identify and quantitate organisms and in some cases viruses. Forexample, a t-RNA probe specific for Legionella can be produced and usedin a manner similar to that described for the R-RNA probe specific onlyfor Legionella. The illustrative embodiment described for the Legionellaspecific R-RNA probe thus also serves as an illustration of a t-RNAspecific Legionella probe.

The genes present in many DNA and RNA viruses include t-RNA genes whichare specific for the virus.

Illustrative Embodiment of the Use of Probes Specific for mRNA, hnRNA,snRNA or psRNA to: Detect, Quantitate and Identify Organisms, Cells andViruses in Cells

Probes specific for mRNA, hnRNA, snRNA or psRNA may be used in a manner;analogous to those for R-RNA and t-RNA to detect, identify andquantitate a specific large or small group of organisms, cells orviruses in cells. Since the evolutionary conservation of the individualgenes which produce those various RNAs varies greatly it is possible toproduce probes which will detect members of very large classes oforganisms and probes which detect members of relatively small classes oforganisms, cells or viruses in cells.

One example of highly conserved gene sequences are the histone genes, afamily of genes present in eukaryotic cells. Histones are nuclearstructural proteins which are present in all eukaryotes. The histone DNAsequences show great similarity even in widely diverged organisms. Thus,the histone genes of sea urchin and man are similar enough to hybridizetogether. A probe which is specific for a particular histone mRNA or fora population of histone mRNAs can detect or identify the presence orabsence of any member of a large group of widely diverse organisms. Thesensitivity of detection of cells or organisms is enhanced by theabundance of the histone mRNAs. In order to grow, a cell or organismmust synthesize histone mRNA in large amounts.

Another example invovles certain gene sequences which code for psRNA andare not conserved during evolution. Such gene sequences from one type oforganism do not hybridize to DNA from distantly related species. A probespecific for one particular psRNA sequence or a population of differentpsRNA sequences from one organism type or virus type, can be used todetect, quantitate, and identify members of a small group of closelyrelated organisms or a small group of closely related viruses which arein cells.

Another example is the developement of a probe specific for a sequenceor sequences of mRNA, hnRNA, snRNA or psRNA which can be used to examinebody fluids for evidence of specific cell damage and destruction. Incertain diseases cells are destroyed and their contents including cellnucleic acids are spilled into the circulating blood. Liver damage dueto hepatitis is one such situation and it is known that both DNA and RNAfrom liver cells enters the circulating blood as a result of celldamage. A probe can be produced which is specific for an RNA sequence orsequences which are characteristic only of liver cells and are notpresent in other normal cell types. The existence of such RNAs is wellknown. This probe can then be used to detect, identify, and quantitateliver specific mRNA, hnRNA, snRNA or psRNA sequences in blood samples bynucleic acid hybridization methodology as described herein. The presenceof liver damage and its extent can be determined since the amount of RNApresent in the blood will reflect the extent of cell damage.

Probes specific for a particular mRNA, hnRNA, snRNA or psRNA sequence orsequences present only in a specific type of liver cell can also beproduced and used to detect and quantitate the presence in the blood ofthe RNA sequences resulting from the damage of a specific liver celltype. Obviously the more abundant the specific RNA sequence in a livercell the higher the sensitivity of detection of the RNA.

Damage to any body tissue or organ (including heart, kidney, brain,muscle, pancreas, spleen, etc.) may be detected and quantitated in thismanner. Other body fluids including spinal fluid and urine can also beassayed for the presence of these specific RNAs.

A useful initial screening test for tissue damage from any source can bedone by utilizing a probe specific for R-RNA and examining blood orother body fluids for the presence of R-RNA or t-RNA sequences.Quantitation of R-RNA or t-RNA present will provide an indication as tothe extent of tissue damage without identifying the source.

Another sample of the use of the nucleic acid hybridization tests andapproaches described herein is the detection and quantitation of E. colicells containing the plasmid genes which code for the E. coli entertoxinprotein. Such a test involves the use of a marked nucleic acid probecomplementary to the enterotoxin protein mRNA in order to detect andquantitate the presence of E. coli bacteria containing the enterotoxinprotein mRNA. This can be accomplished by utilizing the in solutionhybridization methods described herein.

As discussed herein before, the use of a probe complementary to E. colienterotoxin mRNA as a means to detect and quantitate the presence of E.coli bacterial which are producing E. coli enterotoxin and thereforecontain enterotoxin mRNA, by using nucleic acid hybridization methods,has significant advantages over methods such as described in the Falkowet al. patent discussed earlier.

The same approach as described above can be utilized to detect thespecific gene product of a particular microorganism which confersresistance to a particular antibiotic or other antimicrobial agent. Thegenes which confer resistance to most antibiotics are almost alwayspresent on plasmids in the cell. In order for an organism to produce thefactor which confers resistance, the gene for the factor and the mRNAfor the factor must be present in the cell. Thus a probe specific forthe factor mRNA can be used to detect, identify, and quantitate theorganisms which are producing the factor by utilizing nucleic acidhybridization methods.

The above examples of the use of nucleic acid probes specific forparticular sequences or populations of sequences of mRNA, hnRNA, snRNAor psRNA for the purpose of detecting, identifying, and quantitatingparticular groups of organisms, cells, or viruses in cells containingsuch mRNA, hnRNA, snRNA or psRNA sequences, by nucleic acidhybridization methods, are illustrative only, and not limiting.

The Determination of the Sensitivity of Microorganisms toAntimicroorganism Agents

A large number of different clinical situations require thedetermination of antimicrobial agent susceptibility for a variety ofdifferent bacteria and antibiotics (see "Antibiotics in LaboratoryMedicine" by V. Lorian, Editor, Publisher, Williams, and WilkensBaltimore, 1980) All of these situations utilize a method for detectingand quantitating specific classes of microorganisms. In many of thesesituations use of the nucleic acid hybridization tests described earlierwould greatly speed up the determination of antimicrobial agentsusceptibility.

As the organisms in a sample grow and divide, the amount of RNA in theculture increases. A doubling of organisms results in a two foldincrease in the quantity of RNA of different types which is present inthe culture. Thus organism growth can be monitored by determining thequantity of RNA present in the culture at different times after thestart of growth incubation. An increase in the amount of RNA presentwith time indicates organism growth. The magnitude of the increaseindicates the extent of growth. The rate of growth is then the extent ofgrowth per time period. Probes specific for R-RNA, t-RNA, psR-RNA,pst-RNA, certain mRNAs or psmRNAs, certain snRNAs or pssnRNAs, or hnRNAsor pshnRNAs can be used individually or in combination to measure thegrowth of organisms since the quantity of each of these RNAs in aculture will increase as the organisms grow.

A culture of specific category of organisms grown in the presence of anagent or agents which completely inhibit growth will not shown anincrease in RNA with time, while cultures which are partially inhibitedby such agent will show a lower rate of RNA accumulation. A culturewhich is not inhibited will show the same rate of RNA increase as thecontrol culture which does not contain the agent.

One example of this is in determining the susceptibility of Mycobacteriatuberculosis present in a clinical sputum sample. The first step indiagnosing such a sample is to prepare a direct smear of the sputum forstaining in order to detect acid-fast bacilli. It is estimated that itrequires at least 10⁴ -10⁵ M. tuberculosis organisms per ml of sputum toyield a positive direct smear. However, only 10 to 100 of theseorganisms are recoverable by growth culture methods.

If the sputum specimen shows a positive smear, the specimen is thentreated to kill all bacteria except Mycobacteria, and a dilution of thetreated specimen is plated on agar medium containing antimicrobial agentand on control agar which does not contain the agent. Viable individualbacteria will from colonies on the control agar while growth will beinhibited on the agar with the appropriate antimicrobial agent. Theratio of the numbers on the control agar to those on the agent treatedagar is then a measure of the effectiveness of the antimicrobial agent.

A small colony will contain at least 10⁶ bacteria. This means that atleast 20 divisions are needed to form a colony from one bacteria andeach division will take at least 12 hours, for a total of 240 hours or10 days, at a minimum. In most cases it takes 2-4 times this long (3 to6 weeks) for colonies to appear.

A method described earlier for Legionella, would greatly decrease thetime needed for determining antimicrobial agent susceptibility. A probespecific only for R-RNA from members of the genus Mycobacterium could beused in such a test. Such a probe would allow quantitation and adetection sensitivity equal to that described earlier for Legionella. Anucleic acid hybridization test using the accelerated hybridization rateconditions and the excess probe mode of hybridization would easily allowthe detection of about 200 Mycobacteria cells. A step would be added toensure the disruption of the Mycobacteria cells so that the R-RNA wouldbe free to hybridize. Mycobacteria do not readily lyse in the presenceof enzyme-detergent solutions.

As mentioned above, a minimum positive sputum specimen (as determined byacid staining) contains about 10⁴ to 10⁵ Mycobacteria cells per ml andthese 10 to 10² cells can be detected as colony forming units. For drugsusceptibility studies on agar, enough Mycobacteria are added to thecontrol and experimental agar surfaces to ensure that 40 to 50 colonieswill appear on the control agar where no antimicrobial agent is present.If such a practice is followed when using a nucleic acid hybridizationassay this means that the culture is started with about 50 Mycobacteriaand it will then take about 3-4 cell divisions or about 2-3 days inorder to obtain a detectable level of cells. If any significantinhibition of growth by the agent has occurred the control will bepositive and the culture containing agent will be negative. It is clearthat the use of the highly sensitive nucleic acid hybridization methodcan greatly reduce the time needed to determine susceptibility by 5 to10 fold.

The above is just one example of the uses of nucleic acid hybridizationtests such as those described for Legionella for determiningantimicrobial agent sensitivities. The sensitivity of any microorganismcan be determined by utilizing a combination of the standard growthmethodology and an assay for microorganisms based on nucleic acidhybridization. In addition, in many cases the specificity andsensitivity of the nucleic acid hybridization tests for microorganismsallow the determination of antibiotic sensitivity of specific organismseven in the presence of a large excess of other microorganisms oreukaryotic cells.

It is obvious that the same approach can be used to determine thepresence of antimicroorganism activity in blood, urine, other bodyfluids and tissues and other samples. In this case my nucleic acidhybridization procedure can be used to monitor and quantitate the effectof the blood, urine, or other sample on the growth of a specific groupof microorganisms which are put into contact with said blood, using orother samples under conditions where growth occurs if antimicrobialactivity is not present.

A Method for Determining the Growth State of Cells

The overall rate of protein synthesis in a cell is determined by thenumber of ribosomes per cell. The rate of t-RNA synthesis is alsorelated to the number of ribosomes per cell. Inhibition of proteinsynthesis in a cell results in the cessation of R-RNA synthesis by thecells. Indeed, stopping cell growth by any means results in thecessation of R-RNA synthesis and slowing cell growth results in aslowing down of R-RNA synthesis.

The newly synthesized R-RNA molecule is larger than the sum of themature R-RNA subunits present in the ribosome. For example the R-RNA ofE. coli is synthesized as a precursor molecule 6000 bases long. Theprecursor molecule is then processed to yield the R-RNA subunits(totaling about 4500 bases) which are then incorporated into ribosomesand "extra" or precursor specific R-RNA (ps R-RNA) sequences which areeventually degraded by the cell.

R-RNA is not synthesized in non-growing cells and therefore no precursorspecific R-RNA sequences are present in these cells. In this case, largenumbers of R-RNA molecules are present in the cell but no ps R-RNAsequences are present.

In a slowly growing cell a small amount of R-RNA precursor issynthesized and a small amount of psR-RNA is present.

In a rapidly growing cell a large amount of R-RNA precursor issynthesized and several thousand psR-RNA sequences are present.

The absence of psR-RNA in a cell signals that the cell is not growing.The ratio of R-RNA to psR-RNA in a cell is an indication of the growthrate of a cell.

A.timicrobial agents inhibit cell growth. Cells which are not growthinhibited by the agent will contain large amounts of psR-RNA. In cellswhich are only partially growth inhibited the psR-RNA will be present ina loer amount. The ratio of R-RNA to psR-RNA will give a measure of thedegree of inhibition.

A nucleic acid probe specific for the psR-RNA sequences of a particulargroup of microorganisms can be used in a nucleic acid hybridization testto determine and quantitate the presence or absence of psR-RNA in thosemicroorganisms when the organisms are grown in the presence and absenceof a particular antimicroorganism agent or a group of such agents. Thiscan be done even in the presence of large numbers of organisms which arenot related to the microorganism group of interest.

It is obvious that this nucleic acid hybridization method can also beused to determine the presence of substances with antimicroorganismactivity in blood, urine, other body fluids and tissues, and othersamples.

This method of determining growth of cells can be used to determine thestate of growth of any cell which synthesizes R-RNA. The above exampleis only one of many used for such a method. A method based on using aprobe specific for the pst-RNA sequences of a particular group oforganisms or cells can also be used to determine the state of growth ofthose organisms or cells.

A method based on utilizing probes, specific for certain mRNAs, psmRNAs,hnRNAs, pshnRNA, snRNAs, or pssnRNAs, which are abundant in rapidlygrowing organisms or cells but absent, or present in low amount, innon-growing or slow-growing cells can also be used to determine thestate of growth of these organisms or cells. For example, the mRNA for aprotein, RNA polyerase, is present in abundance, several hundred copiesper cell, in rapidly growing cells. In non-growing cells very little RNAis synthesized and little mRNA is present.

A method based on utilizing probe specific for certain virus mRNAs orpsmRNAs which are abundant when said virus is rapidly growing in a celland absent when the virus is present in a cell but not growing, can alsobe used to determine the state of growth of viruses in cells. Thus insituations where members of a particular category of organisms are knownto be present in a sample it is possible to use a single probe todetermine the growth state of said organisms. For example if no ps R-RNAcan be detected in the organisms, they are in a non-growing state. IfpsR-RNA is detected in the organisms but in low amount relative to thenumber of organisms present, the organisms are growing slowly. If largeamounts of psRNA are detected, relative to the number of organismspresent, the organisms are growing rapidly.

Another approach to determining the state of growth of a particularorganism or class of organisms relies on utilizing two probes, each ofwhich will hybridize only to RNA from a particular category of organismsone probe is specific for a stable RNA (R-RNA or t-RNA) which RNA ispresent in said organisms in roughly the same amount in non-growingorganisms or cells and rapidly growing organisms or cells; the otherprobe is specific for a particular mRNA, psmRNA, pst-RNA, pssnRNA,hnRNA, pshnRNA or psR-RNA sequence or sequences which RNA is present inabundance in rapidly growing cells or organisms, absent or present inlow amount in non-growing organisms or cells. These probes are utilizedto detect, identify, and quantitate the amounts present in the sample ofthe RNAs each is specific for. The ratio of the amounts of these RNAs isan indication of the growth state of the organisms or cells.

A specific example of this involves the use of two probes, one specificfor the R-RNA of members of a specific category of organisms or cells,and the other specific for the psR-RNA of the same category of organismsor cells, in order to detect, identify, and quantitate the R-RNA andpsR-RNA present in a sample. The ratio of the amount of psRNA to R-RNApresent in the sample is an indicator of the state of growth of theorganism or cells. In rapidly growing cells there are several thousandcopies of psR-RNA and the psR-RNA/R-RNA ratio is at a maximum. In slowlygrowing cells a relatively small amount of psR-RNA is present and thepsR-RNA/R-RNA ratio is much lower. In non-growing cells psR-RNA shouldbe absent and the psR-RNA/R-RNA ratio is at a minimum.

This same two probe method can be used with a variety of differentcombinations of the probes mentioned above and can be done in thepresence of organisms or cells which are not members of the saidspecific category detected by the probe.

An obvious application of the methods described here to determine thestate of growth of specific categories of organisms is the use of thesemethods to: determine the presence of antimicrobial agents in blood,urine, other body fluids or tissues or other samples; determine thesensitivity of specific categories of organisms to specificantimicrobial agents or groups of such agents. For example bacteriawhich are completely growth inhibited by a particular agent will have aminimum psR-RNA/R-RNA ratio.

Detecting, Identifying, and Quantitating Viruses

It is often important to be able to quickly determine whether aparticular virus or group of viruses is present in a sample. This can bedone by utilizing nucleic acid hybridization tests described herein.

The rapid nucleic acid hybridization test which combines: a) the methodfor rapidly making nucleic acid available for in solution hybridization;b) the method for greatly accelerating the rate of nucleic acidhybridization; c) and the rapid method for assaying for the presence ofhybridized probe; is directly applicable to the detection,identification and quantitation of any group of DNA or RNA virusespresent in a sample by the use of a nucleic acid probe which iscomplementary to the virus group of interest.

In addition, such a virus assay method could be used to determine theeffectiveness of particular antiviral agents and to determine thepresence of antiviral activity in blood, urine and other samples.

Method for Detecting Microorganism Infections by Examining on Organism'sPhagocytic Cells

The extremely high sensitivity and specificity of detectioncharacterizing the nucleic acid hybridization tests specific for R-RNAwhich have been described above, permits of a simple solution to theproblem of obtaining an appropriate clinical specimen for microorganismdiagnosis. A simple blood test sample which contains the white bloodcell (hereinafter referred to as WBC) fraction will suffice in a largenumber of cases.

One manner of using this WBC approach is to first hybridize the WBCsample with a marked probe which will hybridize to R-RNA from any memberof the group of all bacterial but does not hybridize to R-RNA from anyother source. Such a probe serves as a general screening device for anybacteria. Samples which are positive for bacterial R-RNA are thenassayed with a hierarchy of other probes in order to further identifythe bacteria which is present. For example, a probe which hybridizes toR-RNA from any member of the Family Enterbacter but not to R-RNA fromany other source can be used to detect or rule out Enterbacter bacteriawhile a probe specific only for anaerobic R-RNA would be used to detectanaerobes.

The above illustration is just one of may possible ways of using theWBCs as the primary clinical sample for the quick diagnosis ofmicroorganism infections by nucleic acid hybridization. For example,depending on the clinical symptoms of the patient, differentcombinations of probes would be used in order to obtain a diagnosis.

The publications listed below are of interest in connection with variousaspects of the invention and are incorporated herein as part of thedisclosure.

1. Repeated Sequences in DNA R. J. Britten and D. E. Kohne, Science(1968) 161:529.

2. Kinetics of Renaturation of DNA J. G. Wetmur and N. Davidson, J. Mol.Biol. (1968) 31:349.

3. Hydroxyapatite Techniques for Nucleic Acid Reassociation D. E. Kohneand R. J. Britten, in Procedures in Nucleic Acid Research (1971), edsCantoni and Davies, Harper and Row Vol 2:500.

4. Hybridization of Denatured RNA and Small Fragments Transferred toNitrocellulose P. S. Thomas, Proc. Natl. Acad. Sci. USA (1980) 77:5201.

5. DNA-DNA Hybridization on Nitrocellulose Filters: GeneralConsiderations and Non-Ideal Kinetics R. Flavell et al., Wur. J.Biochem. (1974) 47:535.

6. Assay of DNA-RNA Hyrbids by S₁ Nuclease Digestion and Absorption toDEAE-Cellulose Filters In Maxwell et al., Nucleic Acids Research (1978).5:2033.

7. Molecular Cloning: A Laboratory Manual T. Maniatis et al., ColdSpring Harbor Publication (1982).

8. Efficient Transcription of RNA into DNA by Avian Sarcoma VirusPolymerase J. Taylor et al.. Biochemica et Biophys. Acta (1976) 442:324.

9. Use of Specific Radioactive Probes to Stusy Transcription andReplication of the Influenze Virus Genome J. Taylor et al., J. Virology(1977) 21 (2):530.

10. Virus Detection by Nucleic Acid Hybridization: Examination of Normaland ALs Tissue for the Presence of Poliovirus D. Kohne et al., Journalof General Virology (1981) 56:223-233.

11. Leukemogensis by Bovine Leukemia Virus R. Kettmann et al., Proc.Natl. Acad. Sci. USA (1982) 79 (8):2465-2469.

12. Prenatal Diagnosis of a Thalassemia: Clinical Application ofMolecular Hybridization Y. Kan et al., New England Journal of Medicine(1976) 295 (21):1165-1167.

13. Gene Deletions in a Thalassemia Prove that the 5' Locus isFunctional L. Pressley et al., Proc. Natl. Acad, Sci. USA (1980) 77(6):3586-3589.

14. Use of Synthetic Oligonucleotides as Hybridization Probes. S. V.Suggs et al., Proc. Natl. Acad. Sci. USA (1981) 78:6613.

15. Identification of Enterotoxigenic E. coli by Colony HybridizationUsing 3 Enterotoxin Gene Probes S. L. Mosely el atl., J. of Infect.Diseases (1982) 145 (6):863.

16. DNA Reassociation in the Taxonomy of Enteric Bacteria D. Brenner,Int. J. Systematic Bacteriology (1973) 23 (4):298-307.

17. Comparative study of Ribosomal RNA Cistrons in Enterobacteria andMyxobacteria R. Moore et al., J. Bacteriology (1967) 94:1066-1074.

18. Ribosomal RNA Similarities in the Classification of Rhodococcus andRelated Taxa M. Mordarski et al., J. General Microbiology (1980)118:313-319.

19. Retention of Common Nucleotide Sequences in the Ribosomal RNA DNA ofEukaryotes and Some of their Physical Characteristics J. Sinclair etal., Biochemistry (1971) 10:2761.

20. Homologies Among Ribosomal RNA and Messenger RNA Genes inChloroplasts, Mitochondria and E. coli. H. Bohnert et al., Molecular andGeneral Genetics (1980) 179: 539-545.

21. Heterogeneity of the Conserved Ribosomal RNA Sequences of Bacillussubtilis R. Doe et al., J. Bacteriology (1966) 92 (1): 88.

22. Isolation and Characterization of Bacterial Ribosomal RNA CistronsD. Kohne, Biophysical Journal (1968) 8 (10): 1104-1118.

23. Taxonomic Relations Between Archaebacteria 6 Novel Genera Examinedby Cross Hybridization of DNAs and 16S R-RNAs J. Tu et al., J. Mol.Evol. (1982) 18: 109.

24. R-RNA Cistron Homologies Among Hypobomicrobium and Various OtherBacteria, R. Moore, Canadian J. Microbiology (1977) 23: 478.

25. Conservation of Transfer RNA and 5S RNA Cistrons inEnterobacteriaceae D. J. Brenner et al., J. Bacteriology (March 1977)129 (3): 1435.

26. Sequence Homology of Mitochondrial Leucul-tNA Cistron in DifferentOrganisms S. Jakovcic et al., Biochemistry (May 20, 1975) (10) 2037.

27. Synthetic Deoxyligonucleotides as General Probes for Chloroplastt-RNA Genes J. A. Nickoloff and R. B. Hallick, Nucleci Acids Research,(1983) 10 (24): 8191-8210.

28. A.tibiotics in Laboratory Medicine V. Lorian ed, Williams andWilkens (Baltimore/London) 1980.

29. Diagnostic Microbiology Finegold and Martin, Editors, C. V. MosbyCo. (St. Louis) 1982.

30. Spotblot: A Hybridization Assay for Specific DNA Sequences inMultiple Samples M. Cunningham; A.alytical Biochemistry (1983) 415.

31. (29) A.alysis of Repeating DNA Sequences by Reassociation R. Brittenet al., in: Methods in Enzumology XXIX, page 363, Eds. Grossman andMoldave, Academic Press, New York (1974).

32. Studies on Nucleic Acid Reassociation Kinetics: Retarded Rate ofHybridiation of RNA with Excess DNA G. Galau et al., Proc. Natl. Acad.Sci. USA (1974) 74 (6): 2306.

33. Acceleration of DNA Renaturation Rates J. Wetmur, Biopolymers (1975)14: 2517.

34. Room Temperature Method for Increasing the Rate of DNA Reassociationby Many Thousandfold: The Phenol Emulsion Reassociation Technique D.Kohne et al., Biochemistry (1977) 16 (24): 5349.

35. Molecular Biology D. Freifelder, Science Books International(Boston) Van Nostrand Reinhold Co. (New York) 1983.

36. Gene Expression 2 B. Lewin, J. Wiley & Sons, Wiley-IntersciencePublication (1980) New York.

37. Gene Expression 1 B. Lewin, J. Wiley & Sons, Wiley-IntersciencePublication (1974) New York.

As used in the specification and claims the following terms aredefinited as follows:

    ______________________________________                                        DEFINITION OF TERMS                                                           ______________________________________                                        base (see nucleotide)                                                         base pair mismatches                                                          (see imperfectly                                                              complementary base                                                            sequence)                                                                     base sequence, (nucleotide                                                                   A DNA or RNA molecule consisting                               sequence or gene sequence                                                                    of multiple bases.                                             or polynucleotide                                                             sequence or single                                                            strand nucleic acid                                                           sequence)                                                                     complementary base pairs                                                                     Certain of the bases have a chemical                                          affinity for each other and pair                                              together, or are complementary to                                             one another. The complementary                                                base pairs are A:T and G:C in                                                 DNA and A:U in RNA.                                            complementary strands or                                                                     Perfectly complementary nucleic                                complementary base                                                                           acid molecules are nucleic acid                                sequences      molecules in which each base in                                               one molecule is paired with its                                               complementary base in the other                                               strand, to form a stable helical                                              double strand molecule. The                                                   individual strands are termed                                                 complementary strands.                                         criterion      Most preciesely defined as the                                                difference between the temperature                                            of melting of the double strand                                               nucleic acid and the temperature                                              at which hybridization is done.                                               The melting temperature of a                                                  double strand nucleic acid is                                                 determined primarily by the salt                                              concentration of the solution.                                                The criterion determines the degree                                           of complementarity needed for two                                             single strands to form a stable                                               double strand molecules. The                                                  criterion can be described as                                                 highly stringent, or not very                                                 stringent. A highly stringent                                                 criterion requires that two                                                   interacting complementary sequences                                           be highly complementary in sequence                                           in order to form a stable double                                              strand molecule. A poorly stringent                                           criterion is one which allows rela-                                           tively dissimilar complimentary                                               strands to interact and form a                                                double strand molecule. High                                                  stringency allows the presence of                                             only a small fraction of base pair                                            mismatches in a double strand mole-                                           cule. A poorly stringent criterion                                            allows a much larger fraction of                                              base pair mismatches in the hybridi-                                          zation product.                                                denatured or dissociated                                                                     The bond between the paired bases in                           nucleic acid   a double strand nucleic acid molecule                                         can be broken, resulting in two                                               single strand molecules, which                                                then diffuse away from each other.                             double strand nucleic acid                                                                   As it is found in the cell, most DNA                                          is in the double strand state. The                                            DNA is made up of two DNA mole-                                               cules or strands wound helically                                              around each other. The bases face                                             inward and each base is specifi-                                              cally bonded to a complementary                                               base in the other strand. For                                                 example, an A in one strand is                                                always paired with a T in the                                                 other strand, while a G in one                                                strand is paired with a C in                                                  the other strand. In a bacterial                                              cell the double strand molecule is                                            about 5 × 10.sup.6 base pairs long.                                     Each of the bases in one strand of this                                       molecule is paired with its base                                              complement in the other strand. The                                           base sequences of the individual                                              double strand molecules are termed                                            complementary strands.                                         hybridization (see                                                            nucleic and hybridization)                                                    imperfectly complemen-                                                                       Stable double strand molecules can                             tary base sequences (base                                                                    be formed between two strands                                  pair mismatches)                                                                             where a fraction of the bases in the                                          one strand are paired with a non-                                             complementary base in the other                                               strand.                                                        marked probe or marked                                                                       Single strand nucleic acid molecules                           sequence       which are used to detect the presence                                         of other nucleic acids by the process                                         of nucleic acid hybridization. The                                            probe molecules are marked so that                                            they can be specifically detected.                                            This is done by incorporating a                                               specific marker molecule into the                                             nucleic acid or by attaching a                                                specific marker to the nucleic                                                acid. The most effective probes                                               are marked, single strand sequences,                                          which cannot self hybridize but can                                           hybridize only if the nucleic acid                                            to be detected is present. A large                                            number of different markers are                                               available. These include radio-                                               active and fluorescent molecules.                              nucleic acid hybridization                                                                   The bond between the two strands                               or hybridization (reassocia-                                                                 of a double strand molecule can                                tion, or renaturation)                                                                       be broken and the two single strands                                          can be completely separated from                                              each other. Under the proper                                                  conditions the complementary single                                           strands can collide, recognize each                                           other and reform the double strand                                            helical molecule. This process of                                             formation of double strand molecules                                          from complementary single strand                                              molecules is called nucleic acid                                              hybridization.                                                                Nucleic acid hybridization also                                               occurs between partially comple-                                              mentary single strands of RNA and                                             DNA.                                                           nucleotide, nucleotide                                                                       Most DNA consists of sequences of                              base or base   only four nitrogeneous bases:                                                 adenine (A), thymine (T), guanine                                             (G), and cytosine (C). Together                                               these bases form the genetic                                                  alphabet, and long ordered                                                    sequences of them contain, in                                                 coded form, much of the information                                           present in genes.                                                             Most RNA also consists of sequences                                           of only four bases. However, in                                               RNA, thymine is replaced by uridine                                           (U).                                                           reassociation  (see nucleic acid hybridization)                               renaturation   (see nucleic acid hybridization)                               ribosomal RNA or                                                                             The RNA which is present in                                    R-RNA          ribosomes. Virtually all ribosomes                                            contain 3 single strand RNA subunits:                                         one large, one medium-sized, and one                                          small.                                                         ribosome       A cellular particle (containing RNA                                           and protein) necessary for protein                                            synthesis. All life forms except                                              viruses contain ribosomes.                                     R-RNA DNA or   The base sequence in the DNA which                             R-RNA gene     codes for ribosomal RNA. Each                                                 R-RNA subunit is coded for by a                                               separate gene.                                                 R-RNA probe    A marked nucleic acid sequence                                                which is complementary to R-RNA                                               and therefore will hybridize with                                             R-RNA to form a stable double                                                 strand molecule.                                               mRNA           Each individual mRNA is a direct                                              gene product containing the                                                   information necessary to specify a                                            particular protein. The machinery of                                          the cell translates the sequence of the                                       mRNA into a specific protein. Many                                            different mRNAs exist in each cell.                            hnRNA          A complex class of RNA sequences                                              present in the nucleus of eukaryotic                                          cells which includes precursor                                                mRNA molecules. Most hnRNA                                                    sequences never leave the nucleus.                                            The function of most of these                                                 molecules is unknown.                                          snRNA          A class of relatively stable small                                            nuclear RNA molecules which are                                               present primarily in the nucleus of                                           eukaryotic cells in large numbers.                             precursor RNA  Many RNA molecules in both                                                    prokaryotes and eukaryotes are                                                synthesized as part of a large RNA                                            molecules which is then processed to                                          yield mature RNA molecules of                                                 various types and other smaller                                               sequences which are apparently                                                discarded.                                                     precursor specific RNA                                                                       The RNA sequences present in pre-                              (ps RNA)       cursor mRNA, t-RNA, R-RNA,                                                    snRNA, and hnRNA which are not                                                present in the mature R-RNA,                                                  t-RNA, mRNA, snRNA, and hnRNA                                                 molecules.                                                     thermal stability of                                                                         The thermal stability or melting                               double strand nucleic                                                                        temperature at which half of a                                 acid molecules population of double strand molecules                                         has been converted to the single                                              strand form.                                                   restriction enzymes                                                                          Components of the restriction-                                                modification cellular defense                                                 system against foreign nucleic                                                acids. These enzymes cut                                                      unmodified (e.g., methylated)                                                 double-stranded DNA at specific                                               sequences which exhibit twofold                                               symmetry about a point.                                        transfer RNA (t-RNA)                                                                         During protein synthesis individual                                           amino acids are aligned in the proper                                         order by various specific adaptor                                             molecules or t-RNA molecules.                                                 Each amino acid is ordered by a                                               different t-RNA species.                                       ______________________________________                                    

While the invention has been described and illustrated in detail, itwill be apparent to those skilled in the art that various changes,equivalents and alternatives may be resorted to without departing fromthe spirit of the invention, and all of such changes, equivalents andalternatives are contemplated as may come within the scope of theappended claims and equivalents thereof.

What is claimed is:
 1. A method for specifically detecting the presenceor amount of ribosomal nucleic acid in a sample from more than one groupof non-viral organisms, each said group consisting of at least onenon-viral organism, comprising the steps of:a) contacting said samplewith at least two nucleic acid probes, each said probe comprising anucleotide base sequence which hybridizes with only a subsequence ofsaid ribosomal nucleic acid specific to one of said groups of non-viralorganisms under stringent hybridization conditions, and wherein eachsaid nucleotide base sequence does not comprise a sequence whichhybridizes under said conditions to other non-viral organisms; b)incubating said probes and said sample under said conditions, such thateach said nucleotide base sequence hybridizes with said subsequence ofsaid ribosomal nucleic acid specific to one of said groups of non-viralorganisms and not to ribosomal nucleic acids of other non-viralorganisms; and c) assaying for any of said probes hybridized with saidribosomal nucleic acid as an indication of the presence or amount ofsaid ribosomal nucleic acid in said sample.
 2. The method of claim 1,wherein said ribosomal nucleic acid is rRNA.
 3. The method of claim 1,wherein each said group of non-viral organisms belongs to a taxonselected from the group consisting of species, genus, family, order,class, phylum, and kingdom.
 4. The method of claim 1, wherein each saidprobe has a label.
 5. The method of claim 4, wherein said label of eachsaid probe is selected from the group consisting of a radiolabel, afluor, an enzyme and biotin.